Cas9 nucleic acid molecules and their use

ABSTRACT

Described are recombinant nucleic acid molecules for increased expression of Cas9 in human liver. In some embodiments, the recombinant nucleic acid molecules are provided in compositions and methods for gene editing, specifically using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/673,115, filed Aug. 9, 2017, which claims the benefit of U.S.Provisional Application No. 62/372,584, filed Aug. 9, 2016. Theaforementioned applications are incorporated by reference herein intheir entirety.

FIELD

This application pertains to nucleic acid molecules encoding Cas9 andtheir use.

BACKGROUND

The CRISPR-Cas system does not require the generation of customizedproteins to target specific sequences but rather a single Cas enzyme canbe programmed by a short guide RNA molecule to recognize a specific DNAtarget. To utilize the CRISPR-Cas system effectively for genome editingwithout deleterious effects, compositions and methods are needed foroptimization and cell-type/tissue/organ specific delivery of thesegenome engineering tools.

Classic codon optimization introduces synonymous codon substitutions tobetter match the exogenous gene's codon usage to the codon usagepreference of the host. Codon optimization is routinely successful forprokaryotes and unicellular eukaryotes, but has been historically lesssuccessful in multicellular eukaryotes.

Classical codon optimization does not take into account differences incodon usage and tRNA frequencies amongst tissue and cell types. There isa need in the art for tissue specific expression of Cas9 and a Cas9nucleic acid sequence for enhanced expression within a target tissue.

SUMMARY

Disclosed herein are recombinant nucleic acid molecules encoding Cas9that provide increased expression of Cas9 protein in human liver. Inseveral embodiments, the recombinant nucleic acid molecules encodingCas9 are codon-optimized for expression in human liver. The recombinantnucleic acid molecules can be used, for example, for expression of Cas9in the liver, and in CRISPR/Cas9 methods involving gene editing in theliver.

In some embodiments, a recombinant nucleic acid molecule is providedthat comprises a nucleic acid sequence encoding Cas9 that providesincreased expression in human liver compared to native Cas9 sequences.In several embodiments, the recombinant nucleic acid molecule comprisesa nucleotide sequence at least 95% (such as at least 98%, at least 99%,or 100%) identical to SEQ ID NO: 1 or 2. In some embodiments, a vectorincluding the recombinant nucleic acid molecules encoding Cas9 thatprovide increased expression of Cas9 protein in human liver is linked toa promoter. In some embodiments, the vector is an adeno-associated virus(AAV) vector. Methods of making such vectors are also provided.

In some embodiments, an adeno associated virus (AAV) cassette comprisingthe recombinant nucleic acid molecules encoding Cas9 that provideincreased expression of Cas9 protein in human liver. In someembodiments, the nucleic acid molecule is operably linked to a liverspecific promoter (such as a Hepatic Combinatorial Bundle (HCB)promoter), left and right inverted terminal repeats (ITR), and asynthetic polyadenylation (SpA) signal.

In some embodiments, a method is provided for gene-editing liver tissue,including delivering the recombinant nucleic acid molecule encoding Cas9to the liver tissue, and further delivering guide RNAs to the livertissue.

The foregoing and other features and advantages of this disclosure willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the codon frequency of the human genome.

FIG. 1B is an illustration showing that measured levels of tRNA contentvary in both proportion and quantity among cell types.

FIG. 2A-FIG. 2C show comparative heat maps of codon frequencies formouse genome vs human genome (FIG. 2A); hepatocyte vs whole human (FIG.2B); and myeloid vs. whole human (FIG. 2C).

FIG. 3A and FIG. 3B are a graph (FIG. 3A) and table (FIG. 3B)demonstrating the fold difference in codon frequencies of mouse genome,liver, and myeloid cells from human whole genome codon frequencies. Themean fold difference of mouse genome codon frequencies from that of thewhole human genome is 0.046. The mean fold difference of liver codonfrequencies from that of the whole human genome is 0.150. The mean folddifference of myeloid cell codon frequencies from that of the humangenome is 0.135.

FIG. 4 is an illustration of an example Cas9 AAV cassette, including aHepatic Combinatorial Bundle (HCB) promoter, recombinant Cas9 nucleicacid with codon-optimization for expression in human liver, left andright inverted terminal repeats (ITR), and synthetic polyadenylation(SpA) signal.

FIG. 5 is a table displaying the codon usage indices for human liver.Codon frequency and codon count are shown.

FIG. 6 is a table showing the percent sequence identity of the sequencesof SEQ ID NO: 1 and SEQ ID NO: 2 and the starting Cas9 sequence of SEQID NO: 3.

FIG. 7A-FIG. 7C show a series of graphs of liver codon adaptive index(CAI) for the starting Cas9 sequence (FIG. 7A) (SEQ ID NO: 3), arecombinant Cas9 sequence with codon-optimization for expression inhuman liver and including CpG motifs (FIG. 7B) (SEQ ID NO: 1), and arecombinant Cas9 sequence with codon-optimization for expression inhuman liver and without CpG (FIG. 7C) (SEQ ID NO: 2).

FIG. 8A-FIG. 8C show a series of graphs of Frequency of Optimal Codons(FOP) for the starting Cas9 sequence (FIG. 8A) (SEQ ID NO: 3), arecombinant Cas9 sequence with CpG (FIG. 8B) (SEQ ID NO: 1), and arecombinant Cas9 sequence without CpG (FIG. 8C) (SEQ ID NO: 2). The FOPis generated against the custom human liver codon usage index.

FIG. 9A-FIG. 9C show a series of graphs of GC content for the startingCas9 sequence (FIG. 9A) (SEQ ID NO: 3), a recombinant Cas9 sequence withCpG (FIG. 9B) (SEQ ID NO: 1), and a recombinant Cas9 sequence withoutCpG (FIG. 9C) (SEQ ID NO: 2). The ideal percentage range of GC contentis between %. Peaks of % GC content in a 60 bp window have been removed.

FIG. 10 is a table listing the numbers of cis-acting elements andantiviral motifs in the starting Cas9 sequence (SEQ ID NO: 3), arecombinant Cas9 sequence with CpG (SEQ ID NO: 1), and a recombinantCas9 sequence without CpG (SEQ ID NO: 2).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile in the form of the file named “Sequence.txt” (˜49.8 KB), which wascreated on Apr. 27, 2020, and is incorporated by reference herein. Inthe accompanying Sequence Listing:

SEQ ID NO: 1 is a recombinant Cas9 nucleic acid sequence for increasedprotein expression in human liver that contains CpG motifs.

SEQ ID NO: 2 is a recombinant Cas9 nucleic acid sequence without CpGs.

SEQ ID NO: 3 is an exemplary starting Cas9 nucleic acid sequence.

SEQ ID NO: 4 is a recombinant Cas9-NLS-NCG nucleic acid sequence forincreased protein expression in human cells.

SEQ ID NO: 5 is a recombinant Cas9-NLS-WCG Genscript nucleic acidsequence for increased protein expression in human cells.

SEQ ID NO: 6 is a recombinant Cas9-NLS-NCG Genscript nucleic acidsequence for increased protein expression in human liver.

SEQ ID NO: 7 is a recombinant Cas9-NLS-WCG nucleic acid sequence forincreased protein expression in human liver.

SEQ ID NO: 8 is the nucleic acid sequence of the Hepatic CombinatorialBundle, or HCB promoter.

SEQ ID NO: 9 is an amino acid sequence of Cas9.

DETAILED DESCRIPTION I. Overview

Described herein are recombinant nucleic acid molecules for increasedexpression of Cas9 in human liver, and their use. The recombinantnucleic acid molecules can be used in combination with CRISPR fortargeted gene-editing in a tissue specific capacity. The compositionsand methods of the present disclosure can be used in gene therapy forproteins produced in the liver, which can target protein encoding genesthemselves, or genetic regulatory elements, and for understanding liveror liver tissue gene function.

Clinical gene therapy frequently is encumbered by low transgene productbiosynthesis at predictably safe vector doses. It has been hypothesizedthat the presence of rare codons may regulate transgene productexpression through depletion of the available cognate tRNA pool. Codonoptimization is the prominent strategy utilized to overcome thishypothesized limitation and involves replacing rare, presumablytranslation-rate limiting, codons with the more frequent ones. Typicalalgorithms attempt to match the codon usage frequency of the targetorganism's total mRNA pool, which has been shown to approximate theoverall available tRNA concentrations. However, upon closer examination,it appears that both codon frequency and tRNA content vary betweentissue types.

Although human-codon optimized Cas9 sequences have been described, thesesequences are predicted to suffer from limitations for in vivoexpression at least related to tRNA frequencies. Accordingly, providedherein are recombinant nucleic acid molecules encoding Cas9 that provideincreased expression of Cas9 protein in specific human tissues (such asthe liver) compared to native Cas9 sequences.

Also provided is a liver-codon optimized CRISPR (Clustered RegularlyInterspaced Short Palindromic Repeats)/Cas9 (CRISPR associated protein9) system designed for efficient in vivo genome editing. The nativeCRISPR/Cas9 system represents a form of adaptive immunity present inStreptococcus pyrogenes and other bacteria. However, in recent years, ithas become the most utilized tool in the genome editing field and nowprovides the basis for several biotherapeutic approaches as well asgenetically-modified crop and food-producing animals. One suchtherapeutic approach involves the in vivo delivery of Cas9, specificguide RNAs and DNA fragments containing homology to a specific targetregion of the genome as well as some sequence of predicted therapeuticvalue to be inserted into the genome. One barrier to this approach liesin the bacterial nature of the Cas9 sequence, which is not well codonoptimized for expression in humans or other vertebrates.

The liver is a major target for gene therapy, including genome editingapproaches. Thus, a recombinant Cas9 nucleic acid molecule with sequencemodifications to increase expression in liver will enable liver-directedin vivo genome editing.

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Adeno-associated virus (AAV): A small, replication-defective,non-enveloped virus that infects humans and some other primate species.AAV is not known to cause disease and elicits a very mild immuneresponse. Gene therapy vectors that utilize AAV can infect both dividingand quiescent cells and can persist in an extrachromosomal state withoutintegrating into the genome of the host cell. These features make AAV anattractive viral vector for gene therapy. There are currently 11recognized serotypes of AAV (AAV1-11).

Administration/Administer: To provide or give a subject an agent, suchas a therapeutic agent (e.g. a recombinant AAV), by any effective route.Exemplary routes of administration include, but are not limited to,injection (such as subcutaneous, intramuscular, intradermal,intraperitoneal, and intravenous), oral, intraductal, sublingual,rectal, transdermal, intranasal, vaginal and inhalation routes.

Cas9: An RNA-guided RNA endonuclease enzyme that can cut DNA. Cas9 hastwo active cutting sites (HNH and RuvC), one for each strand of thedouble helix. An exemplary native Cas9 protein sequence is shown in SEQID NO: 9.

Cas9 sequences are publicly available. For example, GenBank® AccessionNos. nucleotides 796693 . . . 800799 of CP012045.1 and nucleotides1100046 . . . 1104152 of CP014139.1 disclose Cas9 nucleic acids, andGenBank® Accession Nos. NP_269215.1, AMA70685.1 and AKP81606.1 discloseCas9 proteins. Cas9 is further described in UniProt entry Q99ZW2.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells. cDNA can alsocontain untranslated regions (UTRs) that are responsible fortranslational control in the corresponding RNA molecule.

Complementarity: The ability of a nucleic acid to form hydrogen bond(s)with another nucleic acid sequence by either traditional Watson-Crickbase pairing or other non-traditional types. A percent complementarityindicates the percentage of residues in a nucleic acid molecule whichcan form hydrogen bonds (e.g., Watson-Crick base pairing) with a secondnucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%,70%, 80%, 90%, and 100% complementary). “Perfectly complementary” meansthat all the contiguous residues of a nucleic acid sequence willhydrogen bond with the same number of contiguous residues in a secondnucleic acid sequence. “Substantially complementary” as used hereinrefers to a degree of complementarity that is at least 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, or more nucleotides, or refers to two nucleic acids thathybridize under stringent conditions.

Codon-optimized: A “codon-optimized” nucleic acid refers to a nucleicacid sequence that has been altered such that the codons are optimal forexpression in a particular system (such as a particular species ortissue). For example, a nucleic acid sequence can be optimized forexpression in mammalian cells or in a particular mammalian species (suchas human cells), cell type, or tissue (such as a human liver). Codonoptimization does not alter the amino acid sequence of the encodedprotein.

CpG dinucleotide: DNA base cytosine followed by guanine. Cytosines inCpG dinucleotides can be methylated to form 5-methylcytosine. Thismethylation can alter gene expression.

CRISPR (clustered regularly interspaced short palindromic repeats): DNAloci containing short repetitions of base sequences. Each repetition isfollowed by short segments of “spacer DNA” from previous exposures to avirus. CRISPRs are found in approximately 40% of sequenced bacteriagenomes and 90% of sequenced archaea. CRISPRs are often associated withcas genes that code for proteins related to CRISPRs. The CRISPR/Cassystem is a prokaryotic immune system that confers resistance to foreigngenetic elements such as plasmids and phages and provides a form ofacquired immunity. CRISPR spacers recognize and cut these exogenousgenetic elements in a manner analogous to RNAi in eukaryotic organisms.The CRISPR/Cas system can be used for gene editing (adding, disruptingor changing the sequence of specific genes) and gene regulation. Bydelivering the Cas9 sequence and appropriate guide RNAs into a cell, theorganism's genome can be cut at any desired location.

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine (A), guanine (G), cytosine (C), and thymine (T) bound toa deoxyribose sugar to which a phosphate group is attached. Triplets ofnucleotides (referred to as codons) code for each amino acid in apolypeptide, or for a stop signal. The term codon is also used for thecorresponding (and complementary) sequences of three nucleotides in themRNA into which the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intendedto include the reverse complement of that DNA molecule. Except wheresingle-strandedness is required by the text herein, DNA molecules,though written to depict only a single strand, encompass both strands ofa double-stranded DNA molecule. Thus, a reference to the nucleic acidmolecule that encodes a specific protein, or a fragment thereof,encompasses both the sense strand and its reverse complement. Forinstance, it is appropriate to generate probes or primers from thereverse complement sequence of the disclosed nucleic acid molecules.

Gene: A nucleic acid sequence, typically a DNA sequence, that comprisescontrol and coding sequences necessary for the transcription of an RNA,whether an mRNA or otherwise. For instance, a gene may comprise apromoter, one or more enhancers or silencers, a nucleic acid sequencethat encodes a RNA and/or a polypeptide, downstream regulatory sequencesand, possibly, other nucleic acid sequences involved in regulation ofthe expression of an mRNA.

Most eukaryotic genes contain both exons and introns. The term “exon”refers to a nucleic acid sequence found in genomic DNA that isbioinformatically predicted and/or experimentally confirmed tocontribute a contiguous sequence to a mature mRNA transcript. The term“intron” refers to a nucleic acid sequence found in genomic DNA that ispredicted and/or confirmed not to contribute to a mature mRNAtranscript, but rather to be “spliced out” during processing of thetranscript.

Guide sequence: A polynucleotide sequence having sufficientcomplementarity with a target polynucleotide sequence to hybridize withthe target sequence and direct sequence-specific binding of a Cas9 tothe target sequence. In some examples, the guide sequence is RNA. Insome examples, the guide sequence is DNA. The guide nucleic acid caninclude modified bases or chemical modifications (e.g., see Latorre etal., Angewandte Chemie 55:3548-50, 2016). In some embodiments, thedegree of complementarity between a guide sequence and its correspondingtarget sequence, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT,Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.),SOAP (available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). In some embodiments, a guide sequence is about, orat least, about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotidesin length. In some embodiments, a guide sequence is less than about 75,50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Insome embodiments, a guide sequence is 15-25 nucleotides (such as 18-22or 18 nucleotides).

The ability of a guide sequence to direct sequence-specific binding of aCRISPR complex to a target sequence may be assessed by any suitableassay. For example, the components of a CRISPR system sufficient to forma CRISPR complex, including the guide sequence to be tested, may beprovided to a host cell having the corresponding target sequence, suchas by transfection with vectors encoding the components of the CRISPRsequence, followed by an assessment of preferential cleavage within thetarget sequence, such as by Surveyor assay as described herein.Similarly, cleavage of a target polynucleotide sequence may be evaluatedin a test tube by providing the target sequence, components of a CRISPRcomplex, including the guide sequence to be tested and a control guidesequence different from the test guide sequence, and comparing bindingor rate of cleavage at the target sequence between the test and controlguide sequence reactions.

Homology-directed repair (HDR): A mechanism to repair double strandedDNA lesions. The CRISPR/Cas9 methods disclosed herein, such as thosethat use a disclosed recombinant Cas9 nucleic acid molecule, can be usedfor HDR of one or more target genes, for example during G2 and S phaseof the cell cycle.

Intron: A stretch of DNA within a gene that does not contain codinginformation for a protein. Introns are removed before translation of amessenger RNA.

Inverted terminal repeat (ITR): Symmetrical nucleic acid sequences inthe genome of adeno-associated viruses that promotes efficientreplication. ITR sequences are located at each end of the AAV DNAgenome. The ITRs serve as the origins of replication for viral DNAsynthesis and are essential cis components for generating AAVintegrating vectors.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein, virus or cell) has been substantially separated orpurified away from other biological components in the cell or tissue ofthe organism, or the organism itself, in which the component naturallyoccurs, such as other chromosomal and extrachromosomal DNA and RNA,proteins and cells. Nucleic acid molecules and proteins that have been“isolated” include those purified by standard purification methods. Theterm also embraces nucleic acid molecules and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acid molecules and proteins.

Liver: The liver is an organ found in vertebrates serving a wide rangeof functions, including detoxification, protein synthesis, and theproduction of biochemical utilized in digestions. Liver or liver tissueincludes parenchymal cells commonly referred to as hepatocytes. Liver orLiver tissue can also be liver cells that are non-parenchymal cells,especially as such cells constitute 40% of the total number of livercells even though only 6.5% of its volume; and, examples of suchnon-parenchymal cells liver cells or tissue include sinusoidal hepaticendothelial cells, kupffer cells and hepatic stellate cells. Cells ofthe liver express one or more liver gene product(s).

Modulate: A change in the content of genomic DNA gene. Modulation caninclude, but is not limited to, gene activation (e.g., upregulation),gene repression (e.g., downregulation), gene deletion, polynucleotideinsertion, and/or polynucleotide excision.

Non-homologous end-joining (NHEJ): A mechanism that repairs doublestranded breaks in DNA. The CRISPR/Cas9 methods disclosed herein, suchas those that use a disclosed recombinant Cas9 nucleic acid molecule,can be used for NHEJ of one or more target genes.

Nucleic acid molecule: A polymeric form of nucleotides, which mayinclude both sense and anti-sense strands of RNA, cDNA, genomic DNA, andsynthetic forms and mixed polymers of the above. A nucleotide refers toa ribonucleotide, deoxynucleotide or a modified form of either type ofnucleotide. The term “nucleic acid molecule” as used herein issynonymous with “nucleic acid” and “polynucleotide.” A nucleic acidmolecule is usually at least 10 bases in length, unless otherwisespecified. The term includes single- and double-stranded forms of DNA. Apolynucleotide may include either or both naturally occurring andmodified nucleotides linked together by naturally occurring and/ornon-naturally occurring nucleotide linkages. “cDNA” refers to a DNA thatis complementary or identical to an mRNA, in either single stranded ordouble stranded form. “Encoding” refers to the inherent property ofspecific sequences of nucleotides in a polynucleotide, such as a gene, acDNA, or an mRNA, to serve as templates for synthesis of other polymersand macromolecules in biological processes having either a definedsequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a definedsequence of amino acids and the biological properties resultingtherefrom.

Nucleotide: This term includes, but is not limited to, a monomer thatincludes a base linked to a sugar, such as a pyrimidine, purine orsynthetic analogs thereof, or a base linked to an amino acid, as in apeptide nucleic acid (PNA). A nucleotide is one monomer in apolynucleotide. A nucleotide sequence refers to the sequence of bases ina polynucleotide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids. These sequences are usually translatable into apeptide.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the disclosed vectors.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions (such as vector compositions) to be administered cancontain minor amounts of non-toxic auxiliary substances, such as wettingor emulsifying agents, preservatives, and pH buffering agents and thelike, for example sodium acetate or sorbitan monolaurate. In particularembodiments, suitable for administration to a subject the carrier may besterile, and/or suspended or otherwise contained in a unit dosage formcontaining one or more measured doses of the composition suitable toinduce the desired immune response. It may also be accompanied bymedications for its use for treatment purposes. The unit dosage form maybe, for example, in a sealed vial that contains sterile contents or asyringe for injection into a subject, or lyophilized for subsequentsolubilization and administration or in a solid or controlled releasedosage.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). “Polypeptide” applies to amino acid polymers includingnaturally occurring amino acid polymers and non-naturally occurringamino acid polymer as well as in which one or more amino acid residue isa non-natural amino acid, for example, an artificial chemical mimetic ofa corresponding naturally occurring amino acid. A “residue” refers to anamino acid or amino acid mimetic incorporated in a polypeptide by anamide bond or amide bond mimetic. A polypeptide has an amino terminal(N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide”is used interchangeably with peptide or protein, and is used herein torefer to a polymer of amino acid residues.

Preventing, treating or ameliorating a disease: “Preventing” a disease(such as a liver disorder, or disorder involving proteins expressed inthe liver) refers to inhibiting the full development of a disease.“Treating” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop. “Ameliorating” refers to the reduction in the number orseverity of signs or symptoms of a disease.

Promoter: A region of DNA that directs/initiates transcription of anucleic acid (e.g. a gene). A promoter includes necessary nucleic acidsequences near the start site of transcription. Typically, promoters arelocated near the genes they transcribe. A promoter also optionallyincludes distal enhancer or repressor elements which can be located asmuch as several thousand base pairs from the start site oftranscription. A tissue-specific promoter is a promoter thatdirects/initiated transcription primarily in a single type of tissue orcell. For example, a liver-specific promoter is a promoter thatdirects/initiates transcription in liver tissue to a substantiallygreater extent than other tissue types.

Protein: A biological molecule expressed by a gene or other encodingnucleic acid (e.g., a cDNA) and comprised of amino acids.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purifiedpeptide, protein, virus, vector, or other active compound is one that isisolated in whole or in part from naturally associated proteins andother contaminants. In certain embodiments, the term “substantiallypurified” refers to a peptide, protein, vector, virus or other activecompound that has been isolated from a cell, cell culture medium, orother crude preparation and subjected to fractionation to remove variouscomponents of the initial preparation, such as proteins, cellulardebris, and other components.

Recombinant: A recombinant nucleic acid molecule is one that has asequence that is not naturally occurring, for example, includes one ormore nucleic acid substitutions, deletions or insertions, and/or has asequence that is made by an artificial combination of two otherwiseseparated segments of sequence. This artificial combination can beaccomplished by chemical synthesis or, more commonly, by the artificialmanipulation of isolated segments of nucleic acids, for example, bygenetic engineering techniques.

A recombinant virus is one that includes a genome that includes arecombinant nucleic acid molecule. As used herein, “recombinant AAV”refers to an AAV particle in which a recombinant nucleic acid molecule(such as a recombinant nucleic acid molecule encoding a Cas9 protein)has been packaged.

Response element (RE): A DNA sequence included in a promoter to whichone or more transcription factors can bind to and confer an aspect ofcontrol of gene expression.

Sequence identity: The identity or similarity between two or morenucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Sequencesimilarity can be measured in terms of percentage similarity (whichtakes into account conservative amino acid substitutions); the higherthe percentage, the more similar the sequences are. Homologs ororthologs of nucleic acid or amino acid sequences possess a relativelyhigh degree of sequence identity/similarity when aligned using standardmethods. This homology is more significant when the orthologous proteinsor cDNAs are derived from species which are more closely related (suchas human and mouse sequences), compared to species more distantlyrelated (such as human and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI) and onthe internet, for use in connection with the sequence analysis programsblastp, blastn, blastx, tblastn and tblastx. Additional information canbe found at the NCBI web site.

As used herein, reference to “at least 95% identity” refers to “at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or even100% identity” to a specified reference sequence.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals.

Therapeutically effective amount: A quantity of a specifiedpharmaceutical or therapeutic agent (e.g. a recombinant AAV) sufficientto achieve a desired effect in a subject, or in a cell, being treatedwith the agent. The effective amount of the agent will be dependent onseveral factors, including, but not limited to the subject or cellsbeing treated, and the manner of administration of the therapeuticcomposition.

Vector: A vector is a nucleic acid molecule allowing insertion offoreign nucleic acid without disrupting the ability of the vector toreplicate and/or integrate in a host cell. A vector can include nucleicacid sequences that permit it to replicate in a host cell, such as anorigin of replication. A vector can also include one or more selectablemarker genes and other genetic elements. An expression vector is avector that contains the necessary regulatory sequences to allowtranscription and translation of inserted gene or genes. In someembodiments herein, the vector is an adeno-associated virus (AAV)vector. In some embodiments, the vector is a gamma-retroviral vector, alentiviral vector, or an adenoviral vector. An AAV vector can beproduced by a cassette.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. As used herein, the term “comprises” means“includes.” It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent disclosure, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

III. Recombinant Cas9 Nucleic Acid Molecules

As discussed in the Examples, the cDNA nucleotide sequences coding forCas9 were improved by implementing a codon usage bias specific for thehuman liver cell as compared to naturally occurring nucleotide sequencecoding for the corresponding non-codon optimized sequence for a human.Additional changes were also made to improve translation efficacy, suchas optimization of GC content, mRNA secondary structure, premature PolyAsites, RNA instability motif, stable free energy of mRNA, internal chisites, ribosomal binding sites, cryptic splicing sites, negative CpGislands, SD sequence, TATA boxes, and cyptic terminal signals.

In addition, CpG DNA motifs were removed because they may lead to genemethylation and silencing. Codons were substituted with the most highlyused human/liver alternative that did not result in the formation of a5′-CG-3′ dinucleotide in the sequence. CpG removal can also reduce anyimmune response to a vector including the modified transgene, enhancingthe safety and efficacy of the vector. See J Clin Invest. 2013,123(7):2994-3001, entitled “CpG-depleted adeno-associated virus vectorsevade immune detection.”

A recombinant Cas9 sequence with CpG dinucleotides is provided in SEQ IDNO: 1. A recombinant Cas9 sequence without CpG dinucleotides is providedin SEQ ID NO: 2. In certain embodiments, the recombinant Cas9 sequenceis at least 95%, at least 96%, at least 97%, at least 98%, at least 99%,or at least 99.5% identical to SEQ ID NO: 1 or SEQ ID NO: 2. Asdiscussed in the examples, SEQ ID NOs: 1 and 2 are recombinant Cas9nucleic acid sequences that are codon-optimized for increased expressionof Cas9 in human liver tissue and cells.

SEQ ID NO: 1 and SEQ ID NO: 2 contain nuclear localization sequencesfollowed by a TGA stop codon shown in bolded text below. In certainembodiments, the recombinant Cas9 sequence sequences can be modified toinclude no nuclear localization sequence, or alternative nuclearlocalization sequences, or to include an alternative stop codon. Incertain embodiments, the recombinant Cas9 sequence are at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 without thenuclear localization site or TGA stop codon.

Exemplary nucleic acids can be prepared by cloning techniques, or can begenerated synthetically. Examples of appropriate cloning and sequencingtechniques, and instructions sufficient to direct persons of skillthrough many cloning exercises are known (see, e.g., Sambrook et al.(Molecular Cloning: A Laboratory Manual, 4^(th) ed, Cold Spring Harbor,N.Y., 2012) and Ausubel et al. (In Current Protocols in MolecularBiology, John Wiley & Sons, New York, through supplement 104, 2013).Product information from manufacturers of biological reagents andexperimental equipment also provide useful information. Suchmanufacturers include the SIGMA Chemical Company (Saint Louis, Mo.), R&DSystems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway, N.J.),CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp.,Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCOBRL Life Technologies, Inc. (Gaithersburg, Md.), FlukaChemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),Invitrogen (Carlsbad, Calif.), and Applied Biosystems (Foster City,Calif.).

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

III. Cas9/CRISPR

Clustered regularly interspaced short palindromic repeat (CRISPR)RNA-guided adaptive immune systems that protect bacteria and archaeafrom infection by viruses have been repurposed for genome engineering ina wide variety of cell types and multicellular organisms. CRISPRs areDNA loci containing short repetitions of base sequences. Each repetitionis followed by short segments of spacer DNA from previous exposures to avirus. CRISPRs are often associated with Cas genes. By introducingplasmids containing a Cas gene and specifically constructed CRISPRs intoeukaryotic cells, the eukaryotic genome can be cut at any desiredposition. The Cas9 nuclease for targeted genome editing can includefused nuclear localization signals (NLSs) to a recombinant Cas9 nucleicacid sequence for increased Cas9 expression in human liver. This Cas9sequence can be co-expressed with plasmids expressing the tracrRNA and acrRNA-guide, or a single chimeric guide RNA (gRNA).

Provided herein is a CRISPR/Cas9 system for tissue specific gene editingand gene expression. In an embodiment, the targeted tissue is the humanliver.

A variety of clones containing functionally equivalent nucleic acids canbe constructed, such as nucleic acids which differ in sequence but whichencode the same Cas9 amino acid sequence. Silent mutations in the codingsequence result from the degeneracy (i.e., redundancy) of the geneticcode, whereby more than one codon can encode the same amino acidresidue. Thus, for example, leucine can be encoded by CTT, CTC, CTA,CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, orAGC; asparagine can be encoded by AAT or AAC; aspartic acid can beencoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alaninecan be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAAor CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can beencoded by ATT, ATC, or ATA. Tables showing the standard genetic codecan be found in various sources (see, for example, Stryer, 1988,Biochemistry, 3^(rd) Edition, W.H. 5 Freeman and Co., NY).

Classic codon optimization introduces synonymous codon substitutions tobetter match the exogenous gene's codon usage to the codon usagepreference of the host. This older methodology assumes codon usage biasof the host to be a proxy for steady state tRNA levels (see Quax et al.Mol Cell, 2015, included herein by reference in its entirety.) Codonoptimization is routinely successful for prokaryotes and unicellulareukaryotes, but has been historically less successful in multicellulareukaryotes. Optimization for multicellular organisms has not previouslyconsidered the tissue in which the gene will be expressed.

In contrast, the codon optimization methodology used herein in thecreation of a recombinant Cas9 nucleic acid molecule introducesstructural changes and modifies sequence motifs to optimize mRNA freeenergy, mRNA secondary structure, and RNA instability motifs (e.g.cryptic splice sites, polymerase slippage, etc.)

Based on the genetic code, nucleic acid sequences coding for Cas9 can begenerated. In some examples, such a sequence is optimized for expressionin a host cell, such as a hepatocyte. Codon preferences and codon usagetables for a particular species can be used to engineer recombinant Cas9nucleic acid molecules for protein expression in liver cells or tissue(such as a nucleic acid molecule having at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% sequence identity to SEQID NO: 1 or 2).

In one example, the recombinant Cas9 nucleic acid molecule (such as anucleic acid molecules the at least 95% sequence identity to SEQ ID NO:1 or 2) can be inserted into a vector. In one embodiment, vectors areused for expression in humans. Exemplary promoters for expression inmammalian cells include CMV, EF1a, SV40, PGK1, UBc, human beta actin,CAG, and others. Promoters can be tissue specific, providing enhancedpromoter activity in certain cell types. In an embodiment, a liverspecific promoter is the Hepatic Combinatorial Bundle (HCB) promoter(SEQ ID NO: 8).

The recombinant Cas9 nucleic acid molecule (such as a nucleic acidmolecule with at least 95% sequence identity to SEQ ID NO: 1 or 2) canbe expressed in a variety of liver cell types, including hepaticstellate cells, sinusoidal endothelial cells, phagocytic kuppfer cells,and parenchymal hepatocytes.

The disclosed recombinant Cas9 nucleic acid molecules (such as a nucleicacid molecules the at least 95% sequence identity to SEQ ID NO: 1 or 2),can be used in a CRISPR/Cas9 system to modulate (e.g., increase ordecrease) expression of one or more target genes. Such methods can beperformed in vitro (such as in cell culture), or in vivo (such as in anorganism, embryo, or mammal).

The CRISPR/Cas9 system which utilizes the recombinant Cas9 nucleic acidmolecule (such as a nucleic acid molecule with at least 95% sequenceidentity to SEQ ID NO: 1 or 2) can be used for gene editing in a cell,such as a liver cell. In addition, the recombinant Cas9 nucleic acidmolecules can be used in combination with commercially available kits todesign and develop vectors that include CRISPR/Cas9 genome editingmaterials for manipulating a specific target (e.g., those from Origene,Rockville, Md., from Addgene, Cambridge, Mass., such as the Church LabCRISPR Plasmids, and from Life Technologies, Gaithersburg, Md., such asthe GeneArt® CRISPR Nuclease Vector Kit).

The CRISPR/Cas9 system provided herein typically includes two generalcomponents: (1) the recombinant Cas9 nucleic acid molecule (such as anucleic acid molecule with at least 95% sequence identity to SEQ ID NO:1 or 2), whose expression can be driven by a promoter, such as HCB, and(2) single guide nucleic acid molecule, such as RNA (sgRNA or gRNA),which is operably linked downstream of a target sequence and upstream ofa promoter (such as the HCB promoter). When introduced into cells (forexample as part of a single vector or plasmid or divided into multiplevectors or plasmids), the guide nucleic acid molecule guides the Cas9protein encoded by the recombinant Cas9 nucleic acid molecule to thelocus and Cas9 will cut the target site. Using this system, DNAsequences within the endogenous genome and their functional outputs areeasily edited or modulated.

One or more genes can be targeted by the disclosed methods, such as atleast 1, at least 2, at least 3, at least 4 or at least 5 differentgenes or genetic elements in the organism, such as 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 different genes. In certain embodiments, the targeted genesencode proteins produced in the liver. In certain embodiments, thetargeted genetic elements are regulatory elements controlling expressionof proteins produced in the liver. In one example, the gene isassociated with a liver associated disease or disorder, such as aninherited disease (e.g., hemochromatosis and Alpha-1 AntitrypsinDeficiency, Hemophilia A, hemophilia B.)

IV. Recombinant Vectors and Therapeutic Modalities

Viral vectors can also be prepared that include recombinant Cas9 nucleicacid molecule (such as a nucleic acid molecule with at least 95%sequence identity to SEQ ID NO: 1 or 2). Exemplary viral vectors includepolyoma, SV40, adenovirus, vaccinia virus, adeno-associated virus (AAV),herpes viruses including HSV and EBV, Sindbis viruses, alphaviruses andretroviruses of avian, murine, and human origin. Baculovirus (Autographacalifornica multinuclear polyhedrosis virus; AcMNPV) vectors can be usedand obtained from commercial sources. Other suitable vectors includeretrovirus vectors, orthopox vectors, avipox vectors, fowlpox vectors,capripox vectors, suipox vectors, adenoviral vectors, herpes virusvectors, alpha virus vectors, baculovirus vectors, Sindbis virusvectors, vaccinia virus vectors and poliovirus vectors. Specificexemplary vectors are poxvirus vectors such as vaccinia virus, fowlpoxvirus and a highly attenuated vaccinia virus (MVA), adenovirus,baculovirus and the like. Pox viruses of use include orthopox, suipox,avipox, and capripox virus. Orthopox include vaccinia, ectromelia, andraccoon pox. One example of an orthopox of use is vaccinia. Avipoxincludes fowlpox, canary pox and pigeon pox. Capripox include goatpoxand sheeppox. In one example, the suipox is swinepox. Other viralvectors that can be used include other DNA viruses such as herpes virusand adenoviruses, and RNA viruses such as retroviruses and polio.

The recombinant vectors disclosed herein (for example, a recombinant AAVvector) can be used in several different therapeutic applications,depending on the guide RNAs of the CRISPR/Cas9 system encoded by therecombinant vector(s). Guide RNAs and the recombinant Cas9 nucleic acidmolecule (such as a nucleic acid molecule with at least 95% sequenceidentity to SEQ ID NO: 1 or 2) can be combined on a single cassette orvector, or can be contained on individual vectors. In some embodiments,an additional vector encoding rare, or rate limiting tRNAs, is used intreatment of a disorder relating to proteins expressed in the liver, ora liver disorder in conjunction with the recombinant Cas9 nucleic acidmolecule.

In certain embodiments, a recombinant AAV vector can include a tissuespecific promoter, such a liver specific promoter. An exemplary promoteris HNF1-shortABP-SynO-TSS (also called Hepatic Combinatorial Bundle, orHCB) (SEQ ID NO: 8)

GTTAATCATTAAGTCGTTAATTTTTGTGGCCCTTGCGATGTTTGCTCTGGTTAATAATCTCAGGACAAACAGAGGTTAATAATTTTCCAGATCTCTCTGAGCAATAGTATAAAAGGCCAGCAGCAGCCTGACCACATCTCATCCTC

In certain embodiments, an exemplary AAV cassette has the followingstructure as illustrated in FIG. 4 :

(5′AAV2 ITR)-(HCB Promoter)-(recombinant Cas9 nucleic acidmolecule)-(poly adenylation signal)-(3′AAV2 ITR)

In certain embodiments, recombinant vectors are used in the treatment ofdiseases or disorders related to aberrant proteins, or aberrant proteinexpression of proteins expressed in the liver. In certain embodiments,recombinant vectors are used in the treatment of liver diseases ordisorders, or disorders affecting proteins expressed in the liver. Therecombinant Cas9 nucleic acid molecule (such as a nucleic acid moleculewith at least 95% sequence identity to SEQ ID NO: 1 or 2) and targetguide RNAs encoded by one or more recombinant vectors are administeredto a subject in need thereof for the use in treating a liver disorder,for example a hereditary liver disorder, by CRIPSR gene-editing. Incertain embodiments, guide RNAs can be designed to target aberrant geneexpression contributing to liver disorders or disorders relating toproteins expressed in the liver by targeting the regulatory elements ofknown genes, for example those listed herein. In certain embodiments,guide RNAs can be designed to target abnormalities, for example SNPs,within the genes themselves that are contributing to a liver disorder,or disease related to proteins expressed in the liver. In certainembodiments, multiple guide RNAs targeting the same gene, or regulatoryelements, or different genes and regulatory elements can be used in thetreatment of a subject in need thereof.

In certain embodiments, the uses are for the treatment of hereditaryhemochromatosis (HH), a major disorder of iron overload, Wilson'sdisease, a genetic disorder of copper overload, and alpha1-antitrypsin(al-AT) deficiency. In certain embodiments, the protein is humanAlpha1-antitrypsin (al-AT, Accession: P01009.3), HFE protein (AccessionNP_000401.1 or Q30201), or hepatic protein ATP7B (Accession P35670.4) orvariants with greater than 50, 60, 70, 80, 90, 95, or 95 sequenceidentity or similarity.

In certain embodiments, the uses are for the treatment of Hemophilia Ausing a nucleic acid that encodes a guide RNA targeting Factor VIII(Accession: FN811132.1) or variants with greater than 50, 60, 70, 80,90, 95, or 95 sequence identity or similarity.

In certain embodiments, the uses are for the treatment of Hemophillia Busing a nucleic acid that encodes a guide RNA targeting Factor IX(Accession: K02402.1) or variants with greater than 50, 60, 70, 80, 90,95, or 95 sequence identity or similarity.

In certain embodiments, the use is for the treatment ofhypercholesterolaemia using a nucleic acid that encodes for a guide RNAtargeting human phenylalanine hydroxylase (Accession: P00439.1) orvariants with greater than 50, 60, 70, 80, 90, 95, or 95 sequenceidentity or similarity.

In certain embodiments, the use is for the treatment of Type 1tyrosinemia using a nucleic acid that encodes a guide RNA targetinghuman fumarylacetoacetate hydrolase (Accession: P16930.2) or variantswith greater than 50, 60, 70, 80, 90, 95, or 95 sequence identity orsimilarity.

In certain embodiments, the use is for the treatment of Type 2tyrosinemia using a nucleic acid that encodes for a guide RNA targetinghuman tyrosine aminotransferase (Accession: P17735.1) or variants withgreater than 50, 60, 70, 80, 90, 95, or 95 sequence identity orsimilarity.

In certain embodiments, the use is for the treatment of homocystinuriaand hyperhomocysteinemia using a nucleic acid that encodes a guide RNAtargeting human methylenetetrahydrofolate reductase (Accession:P42898.3) or variants with greater than 50, 60, 70, 80, 90, 95, or 95sequence identity or similarity.

In certain embodiments, the use is for the treatment of hyperlipidemiaand hypercholesterolemia using a nucleic acid that encodes a guide RNAtargeting human medium chain acyl-CoA dehydrogenase (Accession:P11310.1) or variants with greater than 50, 60, 70, 80, 90, 95, or 95sequence identity or similarity.

In certain embodiments, the use is for the treatment of Galactosemiausing a a nucleic acid that encodes a guide RNA targeting humangalactose-1-phosphate uridyl transferase (Accession: P07902.3) orvariants with greater than 50, 60, 70, 80, 90, 95, or 95 sequenceidentity or similarity.

In certain embodiments, the use is for the treatment of Lesch-Nyhansyndrome using a nucleic acid that encodes a guide RNA targeting humanhypoxanthine phosphoribosyl-transferase (Accession: P00492.2) orvariants with greater than 50, 60, 70, 80, 90, 95, or 95 sequenceidentity or similarity.

In certain embodiments, the use is for the treatment of Gaucher diseaseusing a nucleic acid that encodes a guide RNA targeting humancerebrosidase (Accession: P07602.2, Accession: P04062.3) or variantswith greater than 50, 60, 70, 80, 90, 95, or 95 sequence identity orsimilarity.

In certain embodiments, the use is for the treatment of Tay-Sachsdisease using a a nucleic acid that encodes a guide RNA targeting humanbeta-hexosaminidase A (Accession: P06865.2) or variants with greaterthan 50, 60, 70, 80, 90, 95, or 95 sequence identity or similarity.

In certain embodiments, the use is for the treatment of Fabry diseaseusing a nucleic acid that encodes a guide RNA targeting humanα-galactosidase (Accession: P06280.1) or variants with greater than 50,60, 70, 80, 90, 95, or 95 sequence identity or similarity.

In certain embodiments, the use is for the treatment of Hunter syndromeusing a nucleic acid that encodes a guide RNA targeting human iduronatesulphatase (Accession: P22304.1) or variants with greater than 50, 60,70, 80, 90, 95, or 95 sequence identity or similarity.

In certain embodiments, the use is for the treatment of glycogen storagedisease type Ia using a nucleic acid that encodes a guide RNA targetinghuman glucose-6-phosphatase (Accession: P35575.2) or variants withgreater than 50, 60, 70, 80, 90, 95, or 95 sequence identity orsimilarity.

In certain embodiments, the use is for the treatment of ammoniametabolism using a nucleic acid that encodes a guide RNA targeting humanornithine transcarbamylase (Accession: P00480.3) or variants withgreater than 50, 60, 70, 80, 90, 95, or 95 sequence identity orsimilarity.

In certain embodiments, the use is for the treatment of phenylketonuriausing a nucleic acid that encodes a guide RNA targeting humanlow-density lipoprotein receptor (Accession: P01130.1) or variants withgreater than 50, 60, 70, 80, 90, 95, or 95 sequence identity orsimilarity.

In certain embodiments, the use is for the treatment of propionicacidemia using a nucleic acid that encodes a guide RNA targeting humanpropionyl-coenzyme A carboxylase, either PCCA and/or PCCB (Accession:P05166.3 beta, NP_000273.2 alpha, NP_001121164.1 alpha) or variants withgreater than 50, 60, 70, 80, 90, 95, or 95 sequence identity orsimilarity.

Recombinant vectors comprising the recombinant Cas9 nucleic acidmolecule (such as a nucleic acid molecule with at least 95% sequenceidentity to SEQ ID NO: 1 or 2) and/or guide RNAs disclosed herein can bedelivered to the liver via the hepatic artery, the portal vein, orintravenously to yield therapeutic levels of therapeutic proteins,including decreased or increased protein levels, or to correct codingerrors in proteins produced in the liver which result in aberrantproteins. The vector is preferably suspended in a physiologicallycompatible carrier, may be administered to a human or non-humanmammalian patient. Suitable carriers can be selected in view of theindication for which the transfer virus is directed. For example, onesuitable carrier includes saline, which may be formulated with a varietyof buffering solutions (e.g., phosphate buffered saline). Otherexemplary carriers include sterile saline, lactose, sucrose, calciumphosphate, gelatin, dextran, agar, pectin, sesame oil, and water.

Optionally, the compositions of the disclosure may contain otherpharmaceutically acceptable excipients, such as preservatives, orchemical stabilizers. Suitable exemplary preservatives includechlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol, andparachlorophenol. Suitable chemical stabilizers include gelatin andalbumin.

The recombinant viral vectors comprising the recombinant Cas9 nucleicacid molecule (such as a nucleic acid molecule with at least 95%sequence identity to SEQ ID NO: 1 or 2) and/or encoding selected guideRNAs are administered in sufficient amounts to express the recombinantCas9 nucleic acid molecule and selected guide RNAs to provide atherapeutic benefit without undue adverse effects, or with medicallyacceptable physiological effects, which can be determined by thoseskilled in the medical arts. Conventional and pharmaceuticallyacceptable routes of administration include, but are not limited to,direct delivery to a desired organ (e.g., the liver (optionally via thehepatic artery) or lung), oral, inhalation, intranasal, intratracheal,intraarterial, intraocular, intravenous, intramuscular, subcutaneous,intradermal, and other parental routes of administration. Routes ofadministration may be combined, if desired.

Dosages of the recombinant viral vectors will depend primarily onfactors such as the condition being treated, the age, weight and healthof the patient, and may thus vary among patients. For example, atherapeutically effective human dosage of the viral vector is generallyin the range of from about 0.1 ml to about 100 ml of solution containingconcentrations of from about 1×10⁹ to 1×10¹⁶ genomes virus vector.

Recombinant viral vectors of the disclosure provide an efficient geneediting tool and can deliver a selected Cas9 and guide RNA(s) to aselected host cell in vivo or ex vivo. In one embodiment, the vectorsdisclosed herein and the cells are mixed ex vivo; the infected cells arecultured using conventional methodologies; and the transduced cells arere-infused into the patient.

In certain embodiments, recombinant viral vectors are generated incultured cells. A person of ordinary skill in the art would recognizemethods of infecting mammalian cell lines with the recombinant viralvector of the present disclosure and maintain the cells in cultureallowing for translation of the viral genome and replication of thevirus.

V. Kits

Kits are provided that include one or more of the disclosed recombinantCas9 nucleic acid molecules, vectors including such recombinant Cas9nucleic acid molecules, and recombinant cells including such nucleicacid molecules or vectors. In some examples, such components are inseparate vials.

In some examples, the recombinant Cas9 nucleic acid molecule is part ofa vector, such as a plasmid or viral vector. In some examples, therecombinant Cas9 nucleic acid molecule (which may be part of a vector)is present in a cell (such as a bacteria, yeast, or mammalian cell, suchas E. coli). In some examples, the recombinant Cas9 nucleic acidmolecule includes an operably linked promoter, such as HCB, CMV or U6.

In some examples, the disclosed kits further include cell culture orgrowth media, such as media appropriate for growing bacterial, plant,insect, or mammalian cells.

In some examples, the disclosed kits further include a guide nucleicacid molecule (guide RNA) specific for a target nucleic acid molecule,such as a target whose temporal or spatial expression is desired to becontrolled. The guide RNA molecule can be part of a vector, such as aplasmid or viral vector.

EXAMPLES Example 1: Target Specific Codon Optimization

The nucleic acid codons contain redundancies such that distinct codonscan encode the same amino acid. FIG. 1A shows the codon frequenciesacross the human genome. Each codon requires a complimentary tRNA. tRNAcontents vary in both proportion and quantity among cell types (see FIG.1B). Moreover, tRNA content and codon usage vary among tissue types(Dittmar et al. PloS Genet. 2006 December; 2(12):e221. PMID: 17194224.Incorporated herein by reference in its entirety). Just as codonfrequencies vary amongst tissues and cell types, they vary amongorganisms (FIG. 2A-FIG. 2C). Codon frequencies are actually more similarbetween the entire mouse genome and the entire human genome thancomparisons between the codon frequencies of the entire human genomecompared to the codon frequencies of the human liver alone, or humanmyeloid cells alone (FIG. 3 ).

Using this information about the variance in codon and tRNA frequenciesamong organisms, tissues and cell types. Algorithms can be created tocodon-optimize sequences for maximized expression in a specific target.In designing and testing codon-optimization the same algorithm was usedfor whole human, liver, and myeloid optimization. The optimizationallowed for structural and cis-acting sequence motifs to be controlledbetween variants. Utilization of these liver optimized sequences allowsfor interrogation of the impact from codon bias only and for controllingfor structural and sequence motif changes.

Example 2: Recombinant Cas9 Sequences for Improved Expression in HumanTissues

Using an algorithm constructed as described above a recombinant Cas9nucleic acid molecules with a sequence that provides for increasedexpression in liver cells and tissue compared to native Cas9 sequencecan be created. The recombinant Cas9 nucleic acid molecule can bedelivered to human liver tissue, for example by an adeno-associatedvirus (AAV) vector. A cassette for producing such an AAV vector is shownin FIG. 4 which includes a Hepatic Combinatorial Bundle (HCB) promoter,the recombinant Cas9 nucleic acid molecule, left and right invertedterminal repeats (ITR), and synthetic polyadenylation (SpA) signal.

The sequence of the recombinant Cas9 nucleic acid molecule that providesfor increased expression in liver cells and tissue compared to nativeCas9 sequence was created using algorithms which take into considerationcodon usage frequencies for human liver as shown in FIG. 5 . In additionto optimizing sequences for target codon frequencies, sequenceoptimization can include targets for CG content or inclusion orelimination of CpG dinucleotides, consideration of tRNA frequencies,secondary structure and other factors.

For reference, an example of Cas9 protein sequence is provided as SEQ IDNO: 9:

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD

An example recombinant Cas9 sequence that provides for increasedexpression in liver cells and tissue compared to native Cas9 sequence isprovided by SEQ ID NO: 1. The sequence includes CpG dinucleotides andthe Cas9 sequence is followed by a nuclear localization site and stopcodon.

(SEQ ID NO: 1) ATGGACAAGAAGTACTCCATCGGCCTGGACATCGGGACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCTGGGGAACACCGACAGACACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACTCCGGAGAAACCGCTGAGGCTACCCGCCTGAAGAGAACCGCTCGCCGGAGGTACACCAGACGCAAGAACAGGATCTGCTACCTGCAGGAGATCTTCTCCAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGAGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGGAAGAAGCTGGTGGACTCCACCGACAAGGCCGACCTGAGACTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGCGGCCACTTCCTGATCGAGGGGGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCTTCCGGAGTGGACGCTAAGGCTATCCTGAGCGCCAGACTGTCCAAGAGCCGGAGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGGAACCTGATCGCCCTGTCCCTGGGGCTGACCCCCAACTTCAAGAGCAATTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGCGCGTGAACACCGAGATCACCAAGGCCCCCCTGTCCGCCAGCATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGCCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGATACGCTGGATACATCGACGGAGGAGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGACCTGCTGAGGAAGCAGAGAACCTTCGACAACGGCTCCATCCCCCACCAGATCCACCTGGGGGAGCTGCACGCCATCCTGAGACGCCAGGAGGACTTCTACCCCTTCCTGAAGGACAACAGGGAGAAGATCGAGAAGATCCTGACCTTCAGAATCCCATACTACGTGGGACCACTGGCTAGGGGAAACTCCAGATTCGCCTGGATGACCCGGAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGAGCTTCCGCCCAGAGCTTCATCGAGAGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGAGAAAGCCCGCCTTCCTGAGCGGGGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGCAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACTCCGTGGAGATCAGCGGAGTGGAGGACCGCTTCAACGCTTCCCTGGGGACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACCGCGAGATGATCGAGGAGCGGCTGAAGACCTACGCCCACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGCGGAGGAGATACACCGGATGGGGGCGCCTGAGCAGAAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGGAAGACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCAACAGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAAGGAGGACATCCAGAAGGCTCAGGTGTCCGGACAGGGGGACAGCCTGCACGAGCACATCGCTAACCTGGCTGGCAGCCCCGCCATCAAGAAGGGGATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTCATGGGCAGGCACAAGCCCGAGAACATCGTGATCGAGATGGCCAGAGAGAACCAGACCACCCAGAAGGGGCAGAAGAACTCCCGCGAGCGGATGAAGAGGATCGAGGAGGGCATCAAGGAGCTGGGGAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGCGACATGTACGTGGACCAGGAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACCACATCGTGCCCCAGTCCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGCAGCGACAAGAACCGGGGGAAGTCCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCGCCAGCTGCTGAACGCCAAGCTGATCACCCAGCGCAAGTTCGACAACCTGACCAAGGCTGAGAGAGGAGGGCTGTCCGAGCTGGACAAGGCCGGCTTCATCAAGAGGCAGCTGGTGGAAACCAGACAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGAGAACGACAAGCTGATCAGGGAGGTGAAGGTCATCACCCTGAAGTCCAAGCTGGTGAGCGACTTCCGCAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACGACGCTTACCTGAACGCTGTGGTGGGAACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGGGACTACAAGGTGTACGACGTGCGCAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCAGGAAGCGCCCCCTGATCGAAACCAACGGCGAAACCGGAGAGATCGTGTGGGACAAGGGAAGAGACTTCGCTACCGTGCGGAAGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGACCGGCGGGTTCTCCAAGGAGAGCATCCTGCCCAAGAGGAACAGCGACAAGCTGATCGCCAGAAAGAAGGACTGGGACCCCAAGAAGTACGGAGGATTCGACTCCCCAACCGTGGCTTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGTCCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGGATCACCATCATGGAGCGGTCCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGAAGAAAGAGAATGCTGGCTTCCGCCGGAGAGCTGCAGAAGGGAAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCTCCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCTCCGAGTTCAGCAAGCGCGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGTCCGCCTACAACAAGCACAGGGACAAGCCCATCAGAGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGAGCTCCAGCTGCCTTCAAGTACTTCGACACCACCATCGACAGGAAGAGATACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGTCCATCACCGGGCTGTACGAAACCAGAATCGACCTGAGCCAGCTGGGAGGCGACCCCAAGAAGAAGCGCAAGGTGTGA

Another example recombinant Cas9 sequence that provides for increasedexpression in liver cells and tissue compared to native Cas9 sequence isprovided by SEQ ID NO: 2. The sequence does not includes CpGdinucleotides and the Cas9 sequence is followed by a nuclearlocalization site and stop codon.

(SEQ ID NO: 2) ATGGACAAGAAGTACTCCATTGGCCTGGACATTGGGACCAACTCTGTGGGCTGGGCTGTGATCACAGATGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCTGGGGAACACAGACAGACACAGCATCAAGAAGAACCTGATTGGAGCCCTGCTGTTTGACTCTGGAGAAACAGCTGAGGCTACCAGGCTGAAGAGAACAGCTAGGAGGAGATACACCAGAAGAAAGAACAGGATCTGCTACCTGCAGGAGATCTTCTCCAATGAGATGGCCAAGGTGGATGACTCCTTCTTCCACAGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCATGAGAGGCACCCCATCTTTGGCAACATTGTGGATGAGGTGGCCTACCATGAGAAGTACCCCACCATCTACCACCTGAGGAAGAAGCTGGTGGACTCCACAGACAAGGCTGACCTGAGACTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTCCTGATTGAGGGGGACCTGAACCCAGACAACTCTGATGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTTGAGGAGAACCCCATCAATGCTTCTGGAGTGGATGCTAAGGCTATCCTGTCTGCCAGACTGTCCAAGAGCAGGAGGCTGGAGAACCTGATTGCCCAGCTGCCTGGGGAGAAGAAGAATGGCCTGTTTGGGAACCTGATTGCCCTGTCCCTGGGGCTGACCCCCAACTTCAAGAGCAATTTTGACCTGGCTGAGGATGCCAAGCTGCAGCTGTCCAAGGACACCTATGATGATGACCTGGACAACCTGCTGGCCCAGATTGGAGACCAGTATGCTGACCTGTTCCTGGCTGCTAAGAACCTGTCTGATGCCATCCTGCTGTCTGACATCCTGAGGGTGAACACAGAGATCACCAAGGCCCCCCTGTCTGCCAGCATGATCAAGAGATATGATGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGAGGCAGCAGCTGCCTGAGAAGTACAAGGAGATCTTCTTTGACCAGAGCAAGAATGGATATGCTGGATACATTGATGGAGGAGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGATGGCACAGAGGAGCTGCTGGTGAAGCTGAACAGGGAGGACCTGCTGAGGAAGCAGAGAACCTTTGACAATGGCTCCATCCCCCACCAGATCCACCTGGGGGAGCTGCATGCCATCCTGAGAAGACAGGAGGACTTCTACCCCTTCCTGAAGGACAACAGGGAGAAGATTGAGAAGATCCTGACCTTCAGAATCCCATACTATGTGGGACCACTGGCTAGGGGAAACTCCAGATTTGCCTGGATGACCAGGAAGTCTGAGGAAACCATCACCCCCTGGAACTTTGAGGAGGTGGTGGACAAGGGAGCTTCTGCCCAGAGCTTCATTGAGAGGATGACCAACTTTGACAAGAACCTGCCCAATGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTATGAGTACTTCACAGTGTACAATGAGCTGACCAAGGTGAAGTATGTGACAGAGGGCATGAGAAAGCCTGCCTTCCTGTCTGGGGAGCAGAAGAAGGCCATTGTGGACCTGCTGTTCAAGACCAACAGGAAGGTGACAGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATTGAGTGCTTTGACTCTGTGGAGATCTCTGGAGTGGAGGACAGATTCAATGCTTCCCTGGGGACCTACCATGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAATGAGGAGAATGAGGACATCCTGGAGGACATTGTGCTGACCCTGACCCTGTTTGAGGACAGAGAGATGATTGAGGAGAGGCTGAAGACCTATGCCCACCTGTTTGATGACAAGGTCATGAAGCAGCTGAAGAGGAGGAGATACACAGGATGGGGGAGGCTGAGCAGAAAGCTGATCAATGGCATCAGAGACAAGCAGTCTGGGAAGACCATCCTGGACTTCCTGAAGTCTGATGGCTTTGCCAACAGGAACTTCATGCAGCTGATCCATGATGACTCCCTGACCTTCAAGGAGGACATCCAGAAGGCTCAGGTGTCTGGACAGGGGGACAGCCTGCATGAGCACATTGCTAACCTGGCTGGCAGCCCTGCCATCAAGAAGGGGATCCTGCAGACTGTGAAGGTGGTGGATGAGCTGGTGAAGGTCATGGGCAGGCACAAGCCTGAGAACATTGTGATTGAGATGGCCAGAGAGAACCAGACCACCCAGAAGGGGCAGAAGAACTCCAGAGAGAGGATGAAGAGGATTGAGGAGGGCATCAAGGAGCTGGGGAGCCAGATCCTGAAGGAGCACCCTGTGGAGAACACCCAGCTGCAGAATGAGAAGCTGTACCTGTACTACCTGCAGAATGGCAGAGACATGTATGTGGACCAGGAGCTGGACATCAACAGACTGTCTGACTATGATGTGGACCACATTGTGCCCCAGTCCTTCCTGAAGGATGACAGCATTGACAACAAGGTGCTGACCAGATCTGACAAGAATAGGGGGAAGTCTGACAATGTGCCCTCTGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAATGCCAAGCTGATCACCCAGAGAAAGTTTGACAACCTGACCAAGGCTGAGAGAGGAGGGCTGTCTGAGCTGGACAAGGCTGGCTTCATCAAGAGGCAGCTGGTGGAAACCAGACAGATCACCAAGCATGTGGCCCAGATCCTGGACAGCAGGATGAACACCAAGTATGATGAGAATGACAAGCTGATCAGGGAGGTGAAGGTCATCACCCTGAAGTCCAAGCTGGTGTCTGACTTCAGAAAGGACTTCCAGTTCTACAAGGTGAGAGAGATCAACAACTACCACCATGCCCATGATGCTTACCTGAATGCTGTGGTGGGAACAGCCCTGATCAAGAAGTACCCCAAGCTGGAGTCTGAGTTTGTGTATGGGGACTACAAGGTGTATGATGTGAGAAAGATGATTGCCAAGTCTGAGCAGGAGATTGGCAAGGCCACAGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAGATCACCCTGGCCAATGGGGAGATCAGGAAGAGACCCCTGATTGAAACCAATGGGGAAACTGGAGAGATTGTGTGGGACAAGGGAAGAGACTTTGCTACAGTGAGAAAGGTGCTGTCCATGCCCCAGGTGAACATTGTGAAGAAGACAGAGGTGCAGACAGGGGGGTTCTCCAAGGAGAGCATCCTGCCCAAGAGGAACTCTGACAAGCTGATTGCCAGAAAGAAGGACTGGGACCCCAAGAAGTATGGAGGATTTGACTCCCCAACAGTGGCTTACTCTGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGTCCAAGAAGCTGAAGTCTGTGAAGGAGCTGCTGGGGATCACCATCATGGAGAGATCCAGCTTTGAGAAGAACCCCATTGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTTGAGCTGGAGAATGGAAGAAAGAGAATGCTGGCTTCTGCTGGAGAGCTGCAGAAGGGAAATGAGCTGGCCCTGCCCAGCAAGTATGTGAACTTCCTGTACCTGGCCTCCCACTATGAGAAGCTGAAGGGCAGCCCTGAGGACAATGAGCAGAAGCAGCTGTTTGTGGAGCAGCACAAGCACTACCTGGATGAGATCATTGAGCAGATCTCTGAGTTCAGCAAGAGAGTGATCCTGGCTGATGCCAACCTGGACAAGGTGCTGTCTGCCTACAACAAGCACAGGGACAAGCCCATCAGAGAGCAGGCTGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGAGCTCCAGCTGCCTTCAAGTACTTTGACACCACCATTGACAGGAAGAGATACACCAGCACCAAGGAGGTGCTGGATGCCACCCTGATCCACCAGTCCATCACAGGGCTGTATGAAACCAGAATTGACCTGAGCCAGCTGGGAGGAGACCCCAAGAAGAAGAGAAAGGTGTGA.

SEQ ID NO: 1 and SEQ ID NO: 2 contain nuclear localization sequences(GACCCCAAGAAGAAGCGCAAGGTG, nucleotides 4102-4125 of SEQ ID NO: 1)followed by a TGA stop codon shown in bolded text. The recombinant Cas9sequences can be modified to include no nuclear localization sequence,or to include an alternative stop codon.

The percent sequence identity between the starting Cas9 sequence of SEQID NO: 3 and the recombinant Cas9 of SEQ ID NO: 1 is 69%. The percentsequence identity between the starting Cas9 sequence of SEQ ID NO: 3 andthe recombinant Cas9 of SEQ ID NO: 2 is 72%. The percent sequenceidentity between the recombinant Cas9 of SEQ ID NO: 1 and therecombinant Cas9 of SEQ ID NO: 2 is 92% (see FIG. 6 ). An exemplarystarting nucleic acid sequence of Cas9 is shown below with reference toSEQ ID NO: 3. SEQ ID NO: 3 is a human codon optimized Cas9 sequenceavailable from ADDGENE® in plasmid #41815.

An exemplary human codon optimized Cas9 sequence is shown below as SEQID NO: 3.

(SEQ ID NO: 3) ATGGACAAGAAGTACTCCATTGGGCTCGATATCGGCACAAACAGCGTCGGCTGGGCCGTCATTACGGACGAGTACAAGGTGCCGAGCAAAAAATTCAAAGTTCTGGGCAATACCGATCGCCACAGCATAAAGAAGAACCTCATTGGCGCCCTCCTGTTCGACTCCGGGGAGACGGCCGAAGCCACGCGGCTCAAAAGAACAGCACGGCGCAGATATACCCGCAGAAAGAATCGGATCTGCTACCTGCAGGAGATCTTTAGTAATGAGATGGCTAAGGTGGATGACTCTTTCTTCCATAGGCTGGAGGAGTCCTTTTTGGTGGAGGAGGATAAAAAGCACGAGCGCCACCCAATCTTTGGCAATATCGTGGACGAGGTGGCGTACCATGAAAAGTACCCAACCATATATCATCTGAGGAAGAAGCTTGTAGACAGTACTGATAAGGCTGACTTGCGGTTGATCTATCTCGCGCTGGCGCATATGATCAAATTTCGGGGACACTTCCTCATCGAGGGGGACCTGAACCCAGACAACAGCGATGTCGACAAACTCTTTATCCAACTGGTTCAGACTTACAATCAGCTTTTCGAAGAGAACCCGATCAACGCATCCGGAGTTGACGCCAAAGCAATCCTGAGCGCTAGGCTGTCCAAATCCCGGCGGCTCGAAAACCTCATCGCACAGCTCCCTGGGGAGAAGAAGAACGGCCTGTTTGGTAATCTTATCGCCCTGTCACTCGGGCTGACCCCCAACTTTAAATCTAACTTCGACCTGGCCGAAGATGCCAAGCTTCAACTGAGCAAAGACACCTACGATGATGATCTCGACAATCTGCTGGCCCAGATCGGCGACCAGTACGCAGACCTTTTTTTGGCGGCAAAGAACCTGTCAGACGCCATTCTGCTGAGTGATATTCTGCGAGTGAACACGGAGATCACCAAAGCTCCGCTGAGCGCTAGTATGATCAAGCGCTATGATGAGCACCACCAAGACTTGACTTTGCTGAAGGCCCTTGTCAGACAGCAACTGCCTGAGAAGTACAAGGAAATTTTCTTCGATCAGTCTAAAAATGGCTACGCCGGATACATTGACGGCGGAGCAAGCCAGGAGGAATTTTACAAATTTATTAAGCCCATCTTGGAAAAAATGGACGGCACCGAGGAGCTGCTGGTAAAGCTTAACAGAGAAGATCTGTTGCGCAAACAGCGCACTTTCGACAATGGAAGCATCCCCCACCAGATTCACCTGGGCGAACTGCACGCTATCCTCAGGCGGCAAGAGGATTTCTACCCCTTTTTGAAAGATAACAGGGAAAAGATTGAGAAAATCCTCACATTTCGGATACCCTACTATGTAGGCCCCCTCGCCCGGGGAAATTCCAGATTCGCGTGGATGACTCGCAAATCAGAAGAGACCATCACTCCCTGGAACTTCGAGGAAGTCGTGGATAAGGGGGCCTCTGCCCAGTCCTTCATCGAAAGGATGACTAACTTTGATAAAAATCTGCCTAACGAAAAGGTGCTTCCTAAACACTCTCTGCTGTACGAGTACTTCACAGTTTATAACGAGCTCACCAAGGTCAAATACGTCACAGAAGGGATGAGAAAGCCAGCATTCCTGTCTGGAGAGCAGAAGAAAGCTATCGTGGACCTCCTCTTCAAGACGAACCGGAAAGTTACCGTGAAACAGCTCAAAGAAGACTATTTCAAAAAGATTGAATGTTTCGACTCTGTTGAAATCAGCGGAGTGGAGGATCGCTTCAACGCATCCCTGGGAACGTATCACGATCTCCTGAAAATCATTAAAGACAAGGACTTCCTGGACAATGAGGAGAACGAGGACATTCTTGAGGACATTGTCCTCACCCTTACGTTGTTTGAAGATAGGGAGATGATTGAAGAACGCTTGAAAACTTACGCTCATCTCTTCGACGACAAAGTCATGAAACAGCTCAAGAGGCGCCGATATACAGGATGGGGGCGGCTGTCAAGAAAACTGATCAATGGGATCCGAGACAAGCAGAGTGGAAAGACAATCCTGGATTTTCTTAAGTCCGATGGATTTGCCAACCGGAACTTCATGCAGTTGATCCATGATGACTCTCTCACCTTTAAGGAGGACATCCAGAAAGCACAAGTTTCTGGCCAGGGGGACAGTCTTCACGAGCACATCGCTAATCTTGCAGGTAGCCCAGCTATCAAAAAGGGAATACTGCAGACCGTTAAGGTCGTGGATGAACTCGTCAAAGTAATGGGAAGGCATAAGCCCGAGAATATCGTTATCGAGATGGCCCGAGAGAACCAAACTACCCAGAAGGGACAGAAGAACAGTAGGGAAAGGATGAAGAGGATTGAAGAGGGTATAAAAGAACTGGGGTCCCAAATCCTTAAGGAACACCCAGTTGAAAACACCCAGCTTCAGAATGAGAAGCTCTACCTGTACTACCTGCAGAACGGCAGGGACATGTACGTGGATCAGGAACTGGACATCAATCGGCTCTCCGACTACGACGTGGATCATATCGTGCCCCAGTCTTTTCTCAAAGATGATTCTATTGATAATAAAGTGTTGACAAGATCCGATAAAAATAGAGGGAAGAGTGATAACGTCCCCTCAGAAGAAGTTGTCAAGAAAATGAAAAATTATTGGCGGCAGCTGCTGAACGCCAAACTGATCACACAACGGAAGTTCGATAATCTGACTAAGGCTGAACGAGGTGGCCTGTCTGAGTTGGATAAAGCCGGCTTCATCAAAAGGCAGCTTGTTGAGACACGCCAGATCACCAAGCACGTGGCCCAAATTCTCGATTCACGCATGAACACCAAGTACGATGAAAATGACAAACTGATTCGAGAGGTGAAAGTTATTACTCTGAAGTCTAAGCTGGTCTCAGATTTCAGAAAGGACTTTCAGTTTTATAAGGTGAGAGAGATCAACAATTACCACCATGCGCATGATGCCTACCTGAATGCAGTGGTAGGCACTGCACTTATCAAAAAATATCCCAAGCTTGAATCTGAATTTGTTTACGGAGACTATAAAGTGTACGATGTTAGGAAAATGATCGCAAAGTCTGAGCAGGAAATAGGCAAGGCCACCGCTAAGTACTTCTTTTACAGCAATATTATGAATTTTTTCAAGACCGAGATTACACTGGCCAATGGAGAGATTCGGAAGCGACCACTTATCGAAACAAACGGAGAAACAGGAGAAATCGTGTGGGACAAGGGTAGGGATTTCGCGACAGTCCGGAAGGTCCTGTCCATGCCGCAGGTGAACATCGTTAAAAAGACCGAAGTACAGACCGGAGGCTTCTCCAAGGAAAGTATCCTCCCGAAAAGGAACAGCGACAAGCTGATCGCACGCAAAAAAGATTGGGACCCCAAGAAATACGGCGGATTCGATTCTCCTACAGTCGCTTACAGTGTACTGGTTGTGGCCAAAGTGGAGAAAGGGAAGTCTAAAAAACTCAAAAGCGTCAAGGAACTGCTGGGCATCACAATCATGGAGCGATCAAGCTTCGAAAAAAACCCCATCGACTTTCTCGAGGCGAAAGGATATAAAGAGGTCAAAAAAGACCTCATCATTAAGCTTCCCAAGTACTCTCTCTTTGAGCTTGAAAACGGCCGGAAACGAATGCTCGCTAGTGCGGGCGAGCTGCAGAAAGGTAACGAGCTGGCACTGCCCTCTAAATACGTTAATTTCTTGTATCTGGCCAGCCACTATGAAAAGCTCAAAGGGTCTCCCGAAGATAATGAGCAGAAGCAGCTGTTCGTGGAACAACACAAACACTACCTTGATGAGATCATCGAGCAAATAAGCGAATTCTCCAAAAGAGTGATCCTCGCCGACGCTAACCTCGATAAGGTGCTTTCTGCTTACAATAAGCACAGGGATAAGCCCATCAGGGAGCAGGCAGAAAACATTATCCACTTGTTTACTCTGACCAACTTGGGCGCGCCTGCAGCCTTCAAGTACTTCGACACCACCATAGACAGAAAGCGGTACACCTCTACAAAGGAGGTCCTGGACGCCACACTGATTCATCAGTCAATTACGGGGCTCTATGAAACAAGAATCGACCTCTCTCAGCTCGGTGGAGACAGCAGGGCTGACCCCAAGAAGAAGAGGAAGGTGTGA

Sequences can be assessed by codon adaptive index (CAI) for theirdistribution of codon usage frequency along the length of the genesequence. A CAI of 1.0 is considered to be perfect in the desiredexpression tissue, and a CAI of >0.8 is regarded as good, in terms ofhigh gene expression level. The CAI scores and relative frequency percodon position for SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 areshown in FIG. 7A-FIG. 7C.

Sequences can be assessed by their Frequency of Optimal Codons (FOP).The FOP is generated against the custom human liver codon usage index.The percentage distribution of codons in computed codon quality groups.The value of 100 is set for the codon with the highest usage frequencyfor a given amino acid in the desired expression tissue. The FOPs forSEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 are shown in FIG. 8A-8C.

GC content, the percentage of the sequence consisting of guanosine orcytosine is also assessed for optimized sequences. The GC content forSEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 are shown in FIG. 9A-9C.

Example 3: Alternative Optimization Protocols

Alternative optimization protocols exist. Additional codon-optimizedCas9 sequences were also developed. SEQ ID NOs: 4-7 were prepared byGENSCRIPT® algorithms. The utilized algorithm takes into account codonusage bias, GC content, CpG dinucleotides content, mRNA secondarystructure, cryptic splicing sites, premature polyA sites, internal chisites and ribosomal binding sites, negative CpG islands, RNA instabilitymotif (ARE), repeat sequences (direct repeat, reverse repeat and Dyadrepeat), and restriction sites that may interfere with cloning, but donot take into consideration tRNA frequencies.

Human Optimized Cas9-NLS-NCG (SEQ ID NO: 4) was optimized for codonfrequencies similar to that of the whole human genome to have no CpGdinucleotides. SEQ ID NO: 4 has a CAI of 0.93 and a GC content of 49.59.

Human Optimized Cas9-NLS-NCG (SEQ ID NO: 4) is shown below:

(SEQ ID NO: 4) ATGGACAAGAAGTATTCTATTGGCCTGGATATTGGCACAAATTCTGTGGGCTGGGCTGTGATCACAGATGAGTACAAGGTGCCATCTAAGAAGTTTAAGGTGCTGGGCAACACAGATAGGCACAGCATCAAGAAGAATCTGATTGGAGCCCTGCTGTTTGACTCTGGAGAGACAGCAGAGGCAACAAGACTGAAGAGAACAGCCAGAAGAAGGTATACAAGAAGGAAGAATAGGATCTGCTACCTGCAGGAGATCTTCAGCAATGAGATGGCCAAGGTGGATGATTCCTTCTTTCACAGACTGGAGGAGTCTTTCCTGGTGGAGGAGGATAAGAAGCATGAGAGGCACCCCATCTTTGGCAACATTGTGGATGAGGTGGCCTATCATGAGAAGTACCCTACAATCTATCACCTGAGGAAGAAGCTGGTGGACAGCACAGATAAGGCTGACCTGAGACTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTTCTGATTGAGGGAGATCTGAACCCAGACAATTCTGATGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAATCAGCTGTTTGAGGAGAACCCCATCAATGCATCTGGAGTGGATGCAAAGGCAATCCTGTCTGCCAGACTGTCTAAGAGCAGAAGGCTGGAGAACCTGATTGCCCAGCTGCCAGGAGAGAAGAAGAATGGCCTGTTTGGCAATCTGATTGCCCTGAGCCTGGGCCTGACACCCAACTTCAAGTCCAATTTTGATCTGGCAGAGGATGCCAAGCTGCAGCTGAGCAAGGACACCTATGATGATGACCTGGATAACCTGCTGGCCCAGATTGGGGATCAGTATGCTGACCTGTTCCTGGCTGCCAAGAATCTGTCTGATGCCATCCTGCTGTCTGATATCCTGAGAGTGAACACAGAGATCACAAAGGCCCCCCTGTCTGCCTCTATGATCAAGAGGTATGATGAGCACCACCAGGATCTGACCCTGCTGAAGGCCCTGGTGAGACAGCAGCTGCCTGAGAAGTACAAGGAGATCTTCTTTGATCAGTCTAAGAATGGATATGCAGGATATATTGATGGAGGAGCAAGCCAGGAGGAGTTCTACAAGTTTATCAAGCCCATCCTGGAGAAGATGGATGGCACAGAGGAGCTGCTGGTGAAGCTGAATAGGGAGGACCTGCTGAGGAAGCAGAGAACCTTTGATAATGGCTCCATCCCTCACCAGATCCACCTGGGAGAGCTGCATGCAATCCTGAGGAGGCAGGAGGACTTCTACCCATTTCTGAAGGATAACAGGGAGAAGATTGAGAAGATCCTGACATTTAGAATCCCCTACTATGTGGGCCCTCTGGCCAGGGGCAATTCTAGGTTTGCCTGGATGACCAGAAAGTCTGAGGAGACAATCACACCCTGGAACTTTGAGGAGGTGGTGGATAAGGGAGCCTCTGCCCAGTCCTTCATTGAGAGGATGACAAATTTTGACAAGAACCTGCCAAATGAGAAGGTGCTGCCCAAGCACTCTCTGCTGTATGAGTATTTCACAGTGTATAATGAGCTGACAAAGGTGAAGTATGTGACAGAGGGCATGAGAAAGCCTGCCTTCCTGTCTGGAGAGCAGAAGAAGGCCATTGTGGACCTGCTGTTTAAGACCAATAGGAAGGTGACAGTGAAGCAGCTGAAGGAGGACTATTTCAAGAAGATTGAGTGTTTTGATTCTGTGGAGATCTCTGGAGTGGAGGACAGATTCAATGCAAGCCTGGGCACCTACCATGATCTGCTGAAGATCATCAAGGATAAGGACTTCCTGGACAATGAGGAGAATGAGGATATCCTGGAGGACATTGTGCTGACCCTGACACTGTTTGAGGATAGGGAGATGATTGAGGAGAGACTGAAGACATATGCCCACCTGTTTGATGACAAAGTGATGAAGCAGCTGAAGAGAAGGAGATACACTGGATGGGGCAGGCTGTCCAGAAAGCTGATCAATGGCATCAGAGACAAGCAGTCTGGCAAGACAATCCTGGACTTTCTGAAGTCTGATGGCTTTGCCAACAGGAACTTCATGCAGCTGATCCATGATGACTCCCTGACCTTCAAGGAGGATATCCAGAAGGCACAGGTGTCTGGACAGGGAGACTCTCTGCATGAGCACATTGCCAACCTGGCTGGCTCTCCTGCCATCAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGATGAGCTGGTGAAAGTGATGGGCAGGCACAAGCCAGAGAACATTGTGATTGAGATGGCCAGAGAGAATCAGACCACACAGAAGGGCCAGAAGAACAGCAGGGAGAGAATGAAGAGAATTGAGGAGGGCATCAAGGAGCTGGGCTCCCAGATCCTGAAGGAGCACCCTGTGGAGAACACACAGCTGCAGAATGAGAAGCTGTATCTGTACTATCTGCAGAATGGCAGAGATATGTATGTGGACCAGGAGCTGGATATCAACAGACTGTCTGATTATGATGTGGATCACATTGTGCCACAGAGCTTCCTGAAGGATGACTCCATTGACAATAAGGTGCTGACCAGGTCTGACAAGAACAGAGGCAAGTCTGATAATGTGCCCTCAGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGAGGCAGCTGCTGAATGCCAAGCTGATCACACAGAGGAAGTTTGATAACCTGACCAAGGCAGAGAGAGGAGGCCTGTCTGAGCTGGACAAGGCAGGCTTCATCAAGAGGCAGCTGGTGGAGACAAGACAGATCACAAAGCATGTGGCCCAGATCCTGGATTCTAGAATGAACACAAAGTATGATGAGAATGACAAGCTGATCAGGGAGGTGAAAGTGATCACCCTGAAGTCTAAGCTGGTGTCAGACTTTAGGAAGGATTTCCAGTTTTATAAGGTGAGAGAGATCAACAACTACCACCATGCCCATGATGCCTACCTGAATGCTGTGGTGGGCACAGCCCTGATCAAGAAGTACCCTAAGCTGGAGTCTGAGTTTGTGTATGGAGACTATAAGGTGTATGATGTGAGGAAGATGATTGCCAAGTCTGAGCAGGAGATTGGCAAGGCCACAGCCAAGTATTTCTTTTACTCTAACATCATGAATTTCTTTAAGACAGAGATCACACTGGCCAATGGAGAGATCAGGAAGAGACCACTGATTGAGACAAATGGAGAGACAGGAGAGATTGTGTGGGACAAGGGCAGAGATTTTGCCACAGTGAGAAAGGTGCTGAGCATGCCCCAAGTGAATATTGTGAAGAAGACTGAGGTGCAGACAGGAGGCTTCTCTAAGGAGAGCATCCTGCCTAAGAGGAACTCTGATAAGCTGATTGCCAGAAAGAAGGACTGGGATCCTAAGAAGTATGGAGGCTTTGACTCTCCAACAGTGGCCTACTCAGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGTCTAAGAAGCTGAAGTCTGTGAAGGAGCTGCTGGGCATCACCATCATGGAGAGAAGCTCCTTTGAGAAGAATCCTATTGATTTTCTGGAGGCCAAGGGCTATAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCAAAGTACTCCCTGTTTGAGCTGGAGAATGGCAGGAAGAGAATGCTGGCATCTGCTGGAGAGCTGCAGAAGGGCAATGAGCTGGCCCTGCCCAGCAAGTATGTGAACTTCCTGTATCTGGCCTCCCACTATGAGAAGCTGAAGGGCTCCCCTGAGGATAATGAGCAGAAGCAGCTGTTTGTGGAGCAGCACAAGCACTATCTGGATGAGATCATTGAGCAGATCTCAGAGTTCTCTAAGAGAGTGATCCTGGCTGATGCCAATCTGGATAAGGTGCTGAGTGCCTACAACAAGCACAGGGATAAGCCAATCAGAGAGCAGGCAGAGAATATCATCCACCTGTTCACCCTGACAAACCTGGGAGCACCAGCAGCCTTCAAGTATTTTGACACCACAATTGATAGGAAGAGGTACACCTCCACAAAGGAGGTGCTGGATGCCACCCTGATCCACCAGAGCATCACAGGCCTGTATGAGACAAGGATTGACCTGTCCCAGCTGGGAGGAGACCCCAAGAAGAAGAGGAAGGTGTGA

Human Optimized Cas9-NLS-WCG Genscript (SEQ ID NO: 5) was optimized forcodon frequencies similar to that of the whole human genome and containsCpG dinucleotides. SEQ ID NO: 5 has a CAI of 0.94 and a GC content of52.74.

Human Optimized Cas9-NLS-WCG Genscript (SEQ ID NO: 5) is shown below:

(SEQ ID NO: 5) ATGGACAAGAAGTATTCTATCGGCCTGGATATCGGCACAAATAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCATCTAAGAAGTTTAAGGTGCTGGGCAACACCGATCGCCACAGCATCAAGAAGAATCTGATCGGCGCCCTGCTGTTCGACTCCGGAGAGACAGCAGAGGCAACACGGCTGAAGAGAACCGCCCGGAGAAGGTATACACGCCGGAAGAATCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGATTCCTTCTTTCACAGACTGGAGGAGTCTTTCCTGGTGGAGGAGGATAAGAAGCACGAGAGGCACCCCATCTTTGGCAACATCGTGGACGAGGTGGCCTATCACGAGAAGTACCCTACAATCTATCACCTGAGGAAGAAGCTGGTGGACAGCACCGATAAGGCCGACCTGCGCCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTTCTGATCGAGGGCGATCTGAACCCAGACAATTCCGATGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAATCAGCTGTTTGAGGAGAACCCCATCAATGCATCCGGAGTGGACGCAAAGGCAATCCTGTCTGCCAGACTGTCTAAGAGCAGAAGGCTGGAGAACCTGATCGCCCAGCTGCCAGGCGAGAAGAAGAACGGCCTGTTTGGCAATCTGATCGCCCTGAGCCTGGGCCTGACACCCAACTTCAAGTCCAATTTTGATCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTATGACGATGACCTGGATAACCTGCTGGCCCAGATCGGCGATCAGTACGCCGACCTGTTCCTGGCCGCCAAGAATCTGTCCGACGCCATCCTGCTGTCTGATATCCTGAGAGTGAACACCGAGATCACAAAGGCCCCCCTGTCCGCCTCTATGATCAAGCGGTACGACGAGCACCACCAGGATCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCTGAGAAGTACAAGGAGATCTTCTTTGATCAGTCTAAGAATGGATACGCAGGATATATCGACGGAGGAGCAAGCCAGGAGGAGTTCTACAAGTTTATCAAGCCCATCCTGGAGAAGATGGACGGCACAGAGGAGCTGCTGGTGAAGCTGAATAGGGAGGACCTGCTGAGGAAGCAGCGCACCTTTGATAACGGCTCCATCCCTCACCAGATCCACCTGGGAGAGCTGCACGCAATCCTGCGCCGGCAGGAGGACTTCTACCCATTTCTGAAGGATAACAGGGAGAAGATCGAGAAGATCCTGACATTCCGCATCCCCTACTATGTGGGCCCTCTGGCCAGGGGCAATTCTCGCTTTGCCTGGATGACCAGAAAGAGCGAGGAGACAATCACACCCTGGAACTTCGAGGAGGTGGTGGATAAGGGCGCCAGCGCCCAGTCCTTCATCGAGAGGATGACAAATTTTGACAAGAACCTGCCAAATGAGAAGGTGCTGCCCAAGCACTCTCTGCTGTACGAGTATTTCACCGTGTATAACGAGCTGACAAAGGTGAAGTACGTGACCGAGGGCATGCGCAAGCCTGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTTAAGACCAATCGGAAGGTGACAGTGAAGCAGCTGAAGGAGGACTATTTCAAGAAGATCGAGTGTTTTGATAGCGTGGAGATCTCCGGAGTGGAGGGCCGGTTCAACGCAAGCCTGGGCACCTACCACGATCTGCTGAAGATCATCAAGGATAAGGACTTCCTGGACAACGAGGAGAATGAGGATATCCTGGAGGACATCGTGCTGACCCTGACACTGTTTGAGGATCGGGAGATGATCGAGGAGAGACTGAAGACATATGCCCACCTGTTCGATGACAAAGTGATGAAGCAGCTGAAGAGAAGGCGCTACACCGGATGGGGCCGGCTGTCCAGAAAGCTGATCAATGGCATCAGAGACAAGCAGTCCGGCAAGACAATCCTGGACTTTCTGAAGTCTGATGGCTTCGCCAACAGGAACTTCATGCAGCTGATCCACGATGACTCCCTGACCTTCAAGGAGGATATCCAGAAGGCACAGGTGTCCGGACAGGGCGACTCTCTGCACGAGCACATCGCCAACCTGGCCGGCTCTCCTGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAAGTGATGGGCAGGCACAAGCCAGAGAACATCGTGATCGAGATGGCCCGCGAGAATCAGACCACACAGAAGGGCCAGAAGAACAGCCGGGAGAGAATGAAGCGCATCGAGGAGGGCATCAAGGAGCTGGGCTCCCAGATCCTGAAGGAGCACCCTGTGGAGAACACACAGCTGCAGAATGAGAAGCTGTATCTGTACTATCTGCAGAATGGCCGGGATATGTACGTGGACCAGGAGCTGGATATCAACAGACTGTCTGATTACGACGTGGATCACATCGTGCCACAGAGCTTCCTGAAGGATGACTCCATCGACAATAAGGTGCTGACCCGGAGCGACAAGAACAGAGGCAAGAGCGATAATGTGCCCTCCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAATGCCAAGCTGATCACACAGCGGAAGTTTGATAACCTGACCAAGGCAGAGAGAGGAGGCCTGTCCGAGCTGGACAAGGCAGGCTTCATCAAGAGGCAGCTGGTGGAGACACGCCAGATCACAAAGCACGTGGCCCAGATCCTGGATTCTAGAATGAACACAAAGTACGATGAGAATGACAAGCTGATCAGGGAGGTGAAAGTGATCACCCTGAAGTCTAAGCTGGTGAGCGACTTTCGGAAGGATTTCCAGTTTTATAAGGTGAGAGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACAGCCCTGATCAAGAAGTACCCTAAGCTGGAGAGCGAGTTCGTGTACGGCGACTATAAGGTGTACGATGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTATTTCTTTTACTCTAACATCATGAATTTCTTTAAGACCGAGATCACACTGGCCAATGGCGAGATCAGGAAGCGCCCACTGATCGAGACAAACGGCGAGACAGGCGAGATCGTGTGGGACAAGGGCCGGGATTTTGCCACCGTGAGAAAGGTGCTGAGCATGCCCCAAGTGAATATCGTGAAGAAGACCGAGGTGCAGACAGGCGGCTTCTCTAAGGAGAGCATCCTGCCTAAGAGGAACTCCGATAAGCTGATCGCCCGCAAGAAGGACTGGGATCCTAAGAAGTATGGCGGCTTCGACTCTCCAACAGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGTCTAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGAGAAGCTCCTTCGAGAAGAATCCTATCGATTTTCTGGAGGCCAAGGGCTATAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCAAAGTACTCCCTGTTTGAGCTGGAGAACGGCCGGAAGAGAATGCTGGCATCTGCCGGAGAGCTGCAGAAGGGCAATGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTATCTGGCCTCCCACTACGAGAAGCTGAAGGGCTCCCCTGAGGATAACGAGCAGAAGCAGCTGTTTGTGGAGCAGCACAAGCACTATCTGGACGAGATCATCGAGCAGATCTCCGAGTTCTCTAAGAGAGTGATCCTGGCCGACGCCAATCTGGATAAGGTGCTGAGCGCCTACAACAAGCACAGGGATAAGCCAATCCGCGAGCAGGCCGAGAATATCATCCACCTGTTCACCCTGACAAACCTGGGAGCACCAGCAGCCTTCAAGTATTTTGACACCACAATCGATAGGAAGCGGTACACCTCCACAAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACAAGGATCGACCTGTCCCAGCTGGGAGGCGACCCCAAGAAGAAGCGGAAGGTGTGA

Human Liver Optimized Cas9-NLS-NCG Genscript (SEQ ID NO: 6) wasoptimized for codon frequencies similar to that of the human liver andcontains no CpG dinucleotides. SEQ ID NO: 6 has a CAI of 0.89 and a GCcontent of 52.74.

Human Liver Optimized Cas9-NLS-NCG Genscript (SEQ ID NO: 6) is shownbelow:

(SEQ ID NO: 6) ATGGACAAGAAGTATTCTATTGGCCTGGATATTGGCACAAATTCTGTGGGCTGGGCTGTGATCACAGATGAGTACAAGGTGCCATCTAAGAAGTTTAAGGTGCTGGGCAACACAGATAGGCACAGCATCAAGAAGAATCTGATTGGAGCCCTGCTGTTTGACTCTGGAGAGACAGCAGAGGCAACAAGACTGAAGAGAACAGCCAGAAGAAGGTATACAAGAAGGAAGAATAGGATCTGCTACCTGCAGGAGATCTTCAGCAATGAGATGGCCAAGGTGGATGATTCCTTCTTTCACAGACTGGAGGAGTCTTTCCTGGTGGAGGAGGATAAGAAGCATGAGAGGCACCCCATCTTTGGCAACATTGTGGATGAGGTGGCCTATCATGAGAAGTACCCTACAATCTATCACCTGAGGAAGAAGCTGGTGGACAGCACAGATAAGGCTGACCTGAGACTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTTCTGATTGAGGGAGATCTGAACCCAGACAATTCTGATGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAATCAGCTGTTTGAGGAGAACCCCATCAATGCATCTGGAGTGGATGCAAAGGCAATCCTGTCTGCCAGACTGTCTAAGAGCAGAAGGCTGGAGAACCTGATTGCCCAGCTGCCAGGAGAGAAGAAGAATGGCCTGTTTGGCAATCTGATTGCCCTGAGCCTGGGCCTGACACCCAACTTCAAGTCCAATTTTGATCTGGCAGAGGATGCCAAGCTGCAGCTGAGCAAGGACACCTATGATGATGACCTGGATAACCTGCTGGCCCAGATTGGGGATCAGTATGCTGACCTGTTCCTGGCTGCCAAGAATCTGTCTGATGCCATCCTGCTGTCTGATATCCTGAGAGTGAACACAGAGATCACAAAGGCCCCCCTGTCTGCCTCTATGATCAAGAGGTATGATGAGCACCACCAGGATCTGACCCTGCTGAAGGCCCTGGTGAGACAGCAGCTGCCTGAGAAGTACAAGGAGATCTTCTTTGATCAGTCTAAGAATGGATATGCAGGATATATTGATGGAGGAGCAAGCCAGGAGGAGTTCTACAAGTTTATCAAGCCCATCCTGGAGAAGATGGATGGCACAGAGGAGCTGCTGGTGAAGCTGAATAGGGAGGACCTGCTGAGGAAGCAGAGAACCTTTGATAATGGCTCCATCCCTCACCAGATCCACCTGGGAGAGCTGCATGCAATCCTGAGGAGGCAGGAGGACTTCTACCCATTTCTGAAGGATAACAGGGAGAAGATTGAGAAGATCCTGACATTTAGAATCCCCTACTATGTGGGCCCTCTGGCCAGGGGCAATTCTAGGTTTGCCTGGATGACCAGAAAGTCTGAGGAGACAATCACACCCTGGAACTTTGAGGAGGTGGTGGATAAGGGAGCCTCTGCCCAGTCCTTCATTGAGAGGATGACAAATTTTGACAAGAACCTGCCAAATGAGAAGGTGCTGCCCAAGCACTCTCTGCTGTATGAGTATTTCACAGTGTATAATGAGCTGACAAAGGTGAAGTATGTGACAGAGGGCATGAGAAAGCCTGCCTTCCTGTCTGGAGAGCAGAAGAAGGCCATTGTGGACCTGCTGTTTAAGACCAATAGGAAGGTGACAGTGAAGCAGCTGAAGGAGGACTATTTCAAGAAGATTGAGTGTTTTGATTCTGTGGAGATCTCTGGAGTGGAGGACAGATTCAATGCAAGCCTGGGCACCTACCATGATCTGCTGAAGATCATCAAGGATAAGGACTTCCTGGACAATGAGGAGAATGAGGATATCCTGGAGGACATTGTGCTGACCCTGACACTGTTTGAGGATAGGGAGATGATTGAGGAGAGACTGAAGACATATGCCCACCTGTTTGATGACAAAGTGATGAAGCAGCTGAAGAGAAGGAGATACACTGGATGGGGCAGGCTGTCCAGAAAGCTGATCAATGGCATCAGAGACAAGCAGTCTGGCAAGACAATCCTGGACTTTCTGAAGTCTGATGGCTTTGCCAACAGGAACTTCATGCAGCTGATCCATGATGACTCCCTGACCTTCAAGGAGGATATCCAGAAGGCACAGGTGTCTGGACAGGGAGACTCTCTGCATGAGCACATTGCCAACCTGGCTGGCTCTCCTGCCATCAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGATGAGCTGGTGAAAGTGATGGGCAGGCACAAGCCAGAGAACATTGTGATTGAGATGGCCAGAGAGAATCAGACCACACAGAAGGGCCAGAAGAACAGCAGGGAGAGAATGAAGAGAATTGAGGAGGGCATCAAGGAGCTGGGCTCCCAGATCCTGAAGGAGCACCCTGTGGAGAACACACAGCTGCAGAATGAGAAGCTGTATCTGTACTATCTGCAGAATGGCAGAGATATGTATGTGGACCAGGAGCTGGATATCAACAGACTGTCTGATTATGATGTGGATCACATTGTGCCACAGAGCTTCCTGAAGGATGACTCCATTGACAATAAGGTGCTGACCAGGTCTGACAAGAACAGAGGCAAGTCTGATAATGTGCCCTCAGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGAGGCAGCTGCTGAATGCCAAGCTGATCACACAGAGGAAGTTTGATAACCTGACCAAGGCAGAGAGAGGAGGCCTGTCTGAGCTGGACAAGGCAGGCTTCATCAAGAGGCAGCTGGTGGAGACAAGACAGATCACAAAGCATGTGGCCCAGATCCTGGATTCTAGAATGAACACAAAGTATGATGAGAATGACAAGCTGATCAGGGAGGTGAAAGTGATCACCCTGAAGTCTAAGCTGGTGTCAGACTTTAGGAAGGATTTCCAGTTTTATAAGGTGAGAGAGATCAACAACTACCACCATGCCCATGATGCCTACCTGAATGCTGTGGTGGGCACAGCCCTGATCAAGAAGTACCCTAAGCTGGAGTCTGAGTTTGTGTATGGAGACTATAAGGTGTATGATGTGAGGAAGATGATTGCCAAGTCTGAGCAGGAGATTGGCAAGGCCACAGCCAAGTATTTCTTTTACTCTAACATCATGAATTTCTTTAAGACAGAGATCACACTGGCCAATGGAGAGATCAGGAAGAGACCACTGATTGAGACAAATGGAGAGACAGGAGAGATTGTGTGGGACAAGGGCAGAGATTTTGCCACAGTGAGAAAGGTGCTGAGCATGCCCCAAGTGAATATTGTGAAGAAGACTGAGGTGCAGACAGGAGGCTTCTCTAAGGAGAGCATCCTGCCTAAGAGGAACTCTGATAAGCTGATTGCCAGAAAGAAGGACTGGGATCCTAAGAAGTATGGAGGCTTTGACTCTCCAACAGTGGCCTACTCAGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGTCTAAGAAGCTGAAGTCTGTGAAGGAGCTGCTGGGCATCACCATCATGGAGAGAAGCTCCTTTGAGAAGAATCCTATTGATTTTCTGGAGGCCAAGGGCTATAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCAAAGTACTCCCTGTTTGAGCTGGAGAATGGCAGGAAGAGAATGCTGGCATCTGCTGGAGAGCTGCAGAAGGGCAATGAGCTGGCCCTGCCCAGCAAGTATGTGAACTTCCTGTATCTGGCCTCCCACTATGAGAAGCTGAAGGGCTCCCCTGAGGATAATGAGCAGAAGCAGCTGTTTGTGGAGCAGCACAAGCACTATCTGGATGAGATCATTGAGCAGATCTCAGAGTTCTCTAAGAGAGTGATCCTGGCTGATGCCAATCTGGATAAGGTGCTGAGTGCCTACAACAAGCACAGGGATAAGCCAATCAGAGAGCAGGCAGAGAATATCATCCACCTGTTCACCCTGACAAACCTGGGAGCACCAGCAGCCTTCAAGTATTTTGACACCACAATTGATAGGAAGAGGTACACCTCCACAAAGGAGGTGCTGGATGCCACCCTGATCCACCAGAGCATCACAGGCCTGTATGAGACAAGGATTGACCTGTCCCAGCTGGGAGGAGACCCCAAGAAGAAGAGGAAGGTGTGA

Liver Optimized Cas9-NLS-WCG (SEQ ID NO: 7) was optimized for codonfrequencies similar to that of the human liver and contains CpGdinucleotides. SEQ ID NO: 7 has a CAI of 0.96 and a GC content of 58.91.

Liver Optimized Cas9-NLS-WCG (SEQ ID NO: 7) is shown below:

(SEQ ID NO: 7) ATGGACAAGAAGTACTCCATCGGCCTGGACATCGGGACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCTGGGGAACACCGACAGACACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACTCCGGAGAAACCGCTGAGGCTACCCGCCTGAAGAGAACCGCTCGCCGGAGGTACACCAGACGCAAGAACAGGATCTGCTACCTGCAGGAGATCTTCTCCAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGAGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGGAAGAAGCTGGTGGACTCCACCGACAAGGCCGACCTGAGACTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGCGGCCACTTCCTGATCGAGGGGGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCTTCCGGAGTGGACGCTAAGGCTATCCTGAGCGCCAGACTGTCCAAGAGCCGGAGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGGAACCTGATCGCCCTGTCCCTGGGGCTGACCCCCAACTTCAAGAGCAATTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGCGCGTGAACACCGAGATCACCAAGGCCCCCCTGTCCGCCAGCATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGCCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGATACGCTGGATACATCGACGGAGGAGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGACCTGCTGAGGAAGCAGAGAACCTTCGACAACGGCTCCATCCCCCACCAGATCCACCTGGGGGAGCTGCACGCCATCCTGAGACGCCAGGAGGACTTCTACCCCTTCCTGAAGGACAACAGGGAGAAGATCGAGAAGATCCTGACCTTCAGAATCCCATACTACGTGGGACCACTGGCTAGGGGAAACTCCAGATTCGCCTGGATGACCCGGAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGAGCTTCCGCCCAGAGCTTCATCGAGAGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGAGAAAGCCCGCCTTCCTGAGCGGGGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGCAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACTCCGTGGAGATCAGCGGAGTGGAGGACCGCTTCAACGCTTCCCTGGGGACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACCGCGAGATGATCGAGGAGCGGCTGAAGACCTACGCCCACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGCGGAGGAGATACACCGGATGGGGGCGCCTGAGCAGAAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGGAAGACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCAACAGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAAGGAGGACATCCAGAAGGCTCAGGTGTCCGGACAGGGGGACAGCCTGCACGAGCACATCGCTAACCTGGCTGGCAGCCCCGCCATCAAGAAGGGGATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTCATGGGCAGGCACAAGCCCGAGAACATCGTGATCGAGATGGCCAGAGAGAACCAGACCACCCAGAAGGGGCAGAAGAACTCCCGCGAGCGGATGAAGAGGATCGAGGAGGGCATCAAGGAGCTGGGGAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGCGACATGTACGTGGACCAGGAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACCACATCGTGCCCCAGTCCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGCAGCGACAAGAACCGGGGGAAGTCCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCGCCAGCTGCTGAACGCCAAGCTGATCACCCAGCGCAAGTTCGACAACCTGACCAAGGCTGAGAGAGGAGGGCTGTCCGAGCTGGACAAGGCCGGCTTCATCAAGAGGCAGCTGGTGGAAACCAGACAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGAGAACGACAAGCTGATCAGGGAGGTGAAGGTCATCACCCTGAAGTCCAAGCTGGTGAGCGACTTCCGCAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACGACGCTTACCTGAACGCTGTGGTGGGAACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGGGACTACAAGGTGTACGACGTGCGCAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCAGGAAGCGCCCCCTGATCGAAACCAACGGCGAAACCGGAGAGATCGTGTGGGACAAGGGAAGAGACTTCGCTACCGTGCGGAAGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGACCGGCGGGTTCTCCAAGGAGAGCATCCTGCCCAAGAGGAACAGCGACAAGCTGATCGCCAGAAAGAAGGACTGGGACCCCAAGAAGTACGGAGGATTCGACTCCCCAACCGTGGCTTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGTCCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGGATCACCATCATGGAGCGGTCCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGAAGAAAGAGAATGCTGGCTTCCGCCGGAGAGCTGCAGAAGGGAAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCTCCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCTCCGAGTTCAGCAAGCGCGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGTCCGCCTACAACAAGCACAGGGACAAGCCCATCAGAGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGAGCTCCAGCTGCCTTCAAGTACTTCGACACCACCATCGACAGGAAGAGATACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGTCCATCACCGGGCTGTACGAAACCAGAATCGACCTGAGCCAGCTGGGAGGCGACCCCAAGAAGAAGCGCAAGGTGTGA

Example 4: Recombinant AAV Vector for Cas9 Expression

This example illustrates exemplary recombinant AAV vectors encoding aCas9 under control of a variant promoter of reduced length for optimalAAV vector-based protein expression (that is, a genome of 5 kb or fewerbp).

An exemplary AAV cassette with such a sequence is depicted in FIG. 4 .This figure illustrates an AAV cassette for use in the creation of anAAV vector and includes 5′ and 3′ ITRs, the HCB (SEQ ID NO: 8) promoter,a nucleic acid molecule encoding Cas9 such as SEQ ID NO: 1 or SEQ ID NO:2, and a synthetic poly A sequence.

HNF1-shortABP-SynO-TSS (also called Hepatic Combinatorial Bundle, orHCB) (SEQ ID NO: 8)

GTTAATCATTAAGTCGTTAATTTTTGTGGCCCTTGCGATGTTTGCTCTGGTTAATAATCTCAGGACAAACAGAGGTTAATAATTTTCCAGATCTCTCTGAGCAATAGTATAAAAGGCCAGCAGCAGCCTGACCACATCTCATCCTC

An exemplary AAV cassette is shown in FIG. 4 , which has the followingstructure:

(5′AAV2 ITR)-(HCB Promoter)-(recombinant Cas9 nucleic acidmolecule)-(poly adenylation signal)-(3′AAV2 ITR)

Example 5: Treatment Comprising Recombinant Cas9 Molecules

This example describes an exemplary method for the clinical use of thedisclosed recombinant Cas9 sequences SEQ ID NO: 1 and SEQ ID NO: 2.

A patient diagnosed with a disorder affecting proteins produced in theliver, or a liver disorder, such a hemochromatosis and Alpha-1Antitrypsin Deficiency, is selected for treatment. The patient isadministered a therapeutically effective amount of a recombinant AAVcomprising the recombinant Cas9 nucleic acid molecule, such as therecombinant Cas9 of SEQ ID NO: 1 or SEQ ID NO: 2 under control of a HCBpromoter as disclosed herein. The patient is also administered atherapeutically effective amount of a guide RNA targeted to a geneassociated with the diagnosed disorder affecting proteins produced inthe liver, or a liver disorder. The guide RNA can be also be containedon an AAV cassette. The recombinant AAVs can be administeredintravenously. An appropriate therapeutic dose can be selected by amedical practitioner. In some cases, the therapeutically effective doseis in the range of 1×1011 to 1×1014 viral particles (vp)/kg, such asabout 1×1012 vp/kg. In most instances, the patient is administered asingle dose. The health of the subject can be monitored over time todetermine the effectiveness of the treatment.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described embodiments. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

We claim:
 1. A recombinant nucleic acid molecule comprising a nucleotidesequence encoding Cas9, wherein the sequence is at least 95% identicalto SEQ ID NO:
 6. 2. The recombinant nucleic acid molecule of claim 1,wherein the nucleotide sequence is at least 98% identical to SEQ ID NO:6.
 3. The recombinant nucleic acid molecule of claim 1, wherein thenucleotide sequence is at least 99% identical to SEQ ID NO:
 6. 4. Therecombinant nucleic acid molecule of claim 1, wherein the nucleotidesequence is identical to SEQ ID NO:
 6. 5. The recombinant nucleic acidmolecule of claim 1, wherein the nucleotide sequence is codon-optimizedfor expression in human liver.
 6. The recombinant nucleic acid moleculeof claim 1, wherein the nucleotide sequence is isolated.
 7. A vectorcomprising the recombinant nucleic acid molecule of claim 1 operablylinked to a promoter.
 8. The vector of claim 7, wherein the promoter isa Hepatic Combinatorial Bundle promoter comprising a nucleic acidsequence set forth as SEQ ID NO:
 8. 9. The vector of claim 7, whereinthe vector is an adeno-associated virus vector.
 10. An adeno-associatedvirus cassette comprising the recombinant nucleic acid molecule ofclaim
 1. 11. The adeno-associated virus cassette of claim 10, whereinthe nucleic acid molecule is operably linked to a Hepatic CombinatorialBundle promoter, left and right inverted terminal repeats, and asynthetic polyadenylation (SpA) signal.
 12. A method of making arecombinant adeno-associated virus, comprising: introducing theadeno-associated virus vector produced by the cassette of claim 10 intocultured cells; culturing the cells so that the recombinantadeno-associated virus is produced; and purifying the recombinantadeno-associated virus.
 13. A recombinant adeno-associated viruspurified according to the method of claim
 12. 14. A method forgene-editing liver tissue, comprising: delivering and expressing atherapeutically effective amount of the recombinant nucleic acidmolecule encoding Cas9 of claim 1 to the liver tissue; and delivering atherapeutically effective amount of one or more guide RNAs to the livertissue, wherein the one or more guide RNAs hybridize with one or moreendogenous target sequences and direct sequence-specific binding of theCas9 to the endogenous target sequence, wherein the Cas9 cleaves theendogenous target sequence.